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How much of this trust fund portfolio is dedicated to health projects? According to statistics reported by the bank in 2013, about half of the cumulative commitments to financial intermediary funds to date was for the health sector (fig 1). In 2012-13 alone, cash contributions to financial intermediary funds for the health sector totalled around $3.9bn, or 37% of the total cash contributions to bank trust funds.9 The same year, recipient executed trust funds handed out about $430m to the health and social services sector, which represents around 4% of the total disbursements from bank trust funds (fig 2).9 17 Data are not available on bank executed trust fund financing for the health sector, but their relative contribution to the health sector is small as bank executed trust funds for all sectors accounted for just 6% of all trust fund disbursements in 2012-13.9 To put these numbers in perspective, IBRD/IDA lending for core health and social services projects was just over $5bn in 2012-13.18
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other
| 99.8 |
What has driven the bank to increasingly turn to trust funds to finance health projects? Firstly, the flexibility of trust funds allows the bank to raise funds from a diverse group of donors for priority countries, while retaining the bank’s trusted financial management services.6 16 17 Core IBRD/IDA programmes can accept contributions only from governments, but trust funds can accept funding from the private sector. This is especially important for vertical (disease specific) funds: corporations like Exxon Mobil and pharmaceutical companies have contributed to health trust funds, and the Bill and Melinda Gates Foundation was the second largest donor to the recent replenishment of Gavi, the Vaccine Alliance.6 19 New financing mechanisms for many vertical funds also entice donors by allowing them to make programme and budgetary decisions. The Global Fund and Gavi, for example, have their own legal charters and a board of directors on which for-profit private sector representatives have voting power.20 Finally, trust funds can channel funding to countries that are not members of the bank or do not choose to invest in global public goods.6 For instance, the Avian and Human Influenza Facility raised $126m for avian influenza surveillance and control in 2006-13 and allocated some of this funding to “weak link” countries that were not prioritising influenza control interventions.21
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other
| 99.9 |
Secondly, rapid agreement and disbursement of funds allows trust funds to channel the requests of specific donors in the context of specific international events or initiatives.5 6 Donors have explained that they earmark aid because it allows them to respond more quickly to emerging challenges.6 Most trust funds are able to disburse funds more rapidly22 than core IBRD/IDA funding mechanisms because they sidestep traditional bank administrative and operational processes. For example, unlike in core lending, the bank’s board of executive directors usually are not required to approve trust fund proposals.23 Such ability to harness political momentum has been crucial to start up many global health programmes targeting infectious diseases.4 19 20
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| 99.9 |
Thirdly, the narrowly defined goals and measurability of outcomes of trust funded projects make them attractive to donors.6 Trust funds for communicable disease control have increasingly dominated the bank’s trust fund portfolio over the past 15 years (figs 1 and 2).5 12 Some private donors to these funds—particularly the Gates Foundation—have strong preferences for financing technological and disease specific interventions.24 25 The outcomes of these grants are usually measured by simple metrics, like the number of bed nets, vaccines, or drug tablets distributed in specific countries.26 27 Furthermore, because public and private donors to trust funds are often able to earmark their commitments to specific regions or activities, they are able to trace what their aid is buying at the country level.6
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| 99.9 |
Finally, trust funds permit the bank and donors to support innovative regional and global projects that do not fit with the IBRD/IDA country focused allocation system. The bank has highlighted how trust funds allow it to expand its global partnerships for global public goods, emergency response, novel focuses (like gender), and, crucially, the control of communicable diseases.4 10 12 15 Several health trust funds have also allowed the bank to fundraise in new ways or to pilot new financing mechanisms. For instance, the bank has used trust funds to incentivise IDA loans for maternal and child health (see paper 4 of this series on the Global Financing Facility28), to encourage donors to buy down IDA loans for countries investing in polio control,29 30 and to provide Wall Street based insurance against future global pandemics (see paper 5 of this series on the Pandemic Emergency Financing Facility31). Similarly, trust funds supporting health results based financing have enabled the bank to pilot performance based financing at a village level before deciding whether to apply this strategy nationwide.4
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other
| 99.9 |
Several costs, however, emerge from pursuing this model of investment. One major concern is that the bank has become vulnerable to “Trojan multilateralism” or the increased influence of small groups of donors on its health programming.22 32 Indeed, major donors have reported that trust funds are a mechanism for bypassing existing allocation systems and influencing the bank’s priorities.6 This could tilt health funding toward vertical interventions and away from health priorities in the recipient country.22 33 34 For instance, the bank’s Independent Evaluation Group found that the polio buy down programme focused exclusively on providing polio vaccines and not wider health or social services infrastructure4 and that delivery of vertical funds sometimes overburdened weak national health systems.16 Additionally, two key measures that allow IDA to provide performance based allocation for its core projects—the country policy and institutional assessment and worldwide governance indicators—do not apply to trust funds, which further raises the risk that trust funds might not fit the needs of low income countries.3 4
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other
| 99.9 |
A second risk is that trust funds erode capacity of core health, nutrition, and population staff and weaken accountability mechanisms at the bank. While trust funds do not tend to increase the total amount of funding that sovereign donors give to the bank, their separate approval and allocation processes might increase transaction costs for the bank and recipient countries.2 16 This can erode capacity of bank staff to supervise other country based health projects16 and explains why the bank recently began to charge higher, more consistent fees for trust funds.17 35 The bank’s cost recovery framework (charging higher/consistent fees for trust funds) was initiated to prevent capacity erosion—if it charges consistent overhead costs for each trust fund, it can use this income to hire more staff or pay HNP staff directly for their work on trust funds.
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other
| 99.9 |
There is an especially high risk that trust funds that finance global health partnership programmes will lack oversight and accountability. The bank does not have a central unit to oversee its participation in global partnerships,16 and the financial intermediary funds that typically fund these partnerships are not covered under the bank’s standard fiduciary, operational, or administrative policies.36 Environmental controls, overhead fees, and the public’s access to information on financial intermediary funds (FIFs) are therefore variable and not guaranteed.4 10 37 38 Bank executed trust funds also came under fire in 2015, when it was made clear that they do not fall under the mandate of the bank’s inspection panel—the body that countries can turn to if they feel that safeguards have been compromised by a bank project.39 40
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other
| 99.94 |
Finally, the bank’s recent claim that it is a “champion for transparency and accountability”15 in its use of trust funds contrasts with the reality of trust fund data and operations. The bank emphasises its efforts to “reform the trust funds framework” by “defining mobilisation objectives more strategically, simplifying and harmonizing agreements, improving cost recovery, and incorporating these funds more fully into the budgetary allocation process.”41 It points to the availability of financial and non-financial information about trust funds through AidFlows, the Financial Intermediary Fund Trustee website, and the World Bank Finances platform.4 15 These resources, however, have major transparency problems for members of the public and researchers (fig 3). Figure 3 suggests specific ways in which these problems could be dealt with.
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other
| 99.9 |
The health financing landscape has transformed over the past 30 years as private aid flows increasingly overtake official development assistance.42 43 With this shift, the supremacy of IBRD’s non-concessional financing model has ended, and IDA, IFC, and trust fund commitments now dwarf its own.6 At the same time, the bank has transformed from a country based lender to a development organisation with representation on the most global partnership programmes in the world.16
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other
| 99.94 |
Trust funds reflect the World Bank’s eagerness to capitalise on these private aid flows for global health activities. This business decision largely explains the proliferation of ad hoc administrative and operational policies for IBRD/IDA trust funds and financial intermediary funds. Competitive interests of private investors to IFC also explain its restrictive access to information policy44 and customised policies about safeguards. In our view, however, trust funds have operated largely in the shadows and beyond the purview of members of the public, without having to conform to the measures taken to increase monitoring and accountability in the bank’s core work. We call on the bank to commit to its “Forward Look” strategy41 for a stronger World Bank Group by improving its trust fund transparency (fig 3 gives explicit recommendations) as a first step.
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other
| 99.94 |
The Bank has four major types of trust funds: IBRD/IDA bank executed trust funds, IBRD/IDA recipient executed trust funds, financial intermediary funds, and IFC trust funds. These funds have distinct purposes, implementation mechanisms, and accountability frameworks
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other
| 99.94 |
Drugs that are used to suppress antiallograft immunity in kidney transplant recipients (KTR) are nephrotoxic and increases the incidence of malignancy and cardiovascular pathology.1 Investigations are underway to use immune regulatory cellular therapy to induce immunological unresponsiveness to donor alloantigens,2 which may permit a reduction of immunosuppression in the longer term. Infusion of Treg cells is 1 proposed cellular therapy undergoing investigation in KTRs.3 Regulatory T cells infused into transplant patients must be able to survive, retain suppressive function, and potentially continue to expand to replace Treg cells that die as part of natural cell turnover in the presence of immunosuppressants, including calcineurin inhibitors (CNIs), such as tacrolimus (TAC). Regulatory T cells require low doses of IL-2 to expand and become functionally active.4,5 CNIs interfere with IL-2 signaling and have been implicated to reduce Treg numbers posttransplant.6,7 The ONE study clinical trial is examining Treg cell adoptive transfer therapy in combination with withdrawal of steroids and mycophenolate mofetil immunosuppression in KTRs; however, the withdrawal of TAC therapy is not currently planned and therefore the identification of Treg populations that remain suppressive in the presence of TAC may be critical to implementing Treg cell therapy successfully in an allograft transplant setting.
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review
| 99.9 |
Low or absent CD127 cell surface expression in combination with CD25 expression is an important and long-established marker to aid the distinction of Treg from T effector (Teff) cells that express higher levels of CD127 in humans. Forkhead box protein (FOX)3 expression inversely correlates with CD127 expression and has been shown to directly regulate CD127 expression through binding the CD127 promoter.8,9 Human CD25+CD127−/lo Treg cells have been demonstrated to be highly suppressive in vivo in a humanized mouse model.10,11 Live human CD25+CD127−/lo Treg cells may be purified and subsequently validated as bona fide Treg by ensuring a high enrichment of FOXP3+ cells and demethylation of the Treg cell-specific demethylated region (TSDR) of the FOXP3 promoter.12 The latter finding is important because downregulation of CD127 and upregulation CD25 and FOXP3 expression by human activated conventional CD4+ T cells are possible.12 Distinct populations within total CD4+ T cells have also been identified using CD25 and CD45RA cell surface markers, each with differential FOXP3 expression and suppressive capacity.13,14 Here we demonstrate that CD4+CD25+CD127−/lo Treg sorted according to differential CD45RA expression distinguishes Treg subpopulations that, after in vitro expansion with a physiological concentration of TAC, have a differential TSDR demethylated phenotype and suppressive function. These observations will inform the design of protocols to deliver Treg cellular therapy to transplant patients receiving CNIs, including TAC.
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study
| 100.0 |
Cells were stained with 7-AAD viability dye, anti-CD3 eFluor450, anti-CD4 PE-eFluor 647 (eBioscience), anti-CD4 electron coupled dye (Beckmann Coulter), anti-CD4 fluorescein isothiocyanate (FITC), anti-CD3 PE, anti-CD3 APC-Cy7, anti-CD8 PE, anti-CD8 APC-Cy7, anti-CD25 PECy7, anti-CD127 PE, and anti-CD27 FITC (BD), anti-CD45RA FITC, FOXP3 Alexa Fluor 647 (BioLegend) specific antibodies. FOXP3 intracellular staining was performed using FOXP3 staining buffers (eBioscience). Data were acquired using a fluorescence activated cell sorting (FACS)CantoII and analysed using FACSDiva software (BD) and Flojo software (Treestar).
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study
| 99.9 |
Peripheral blood mononuclear cells (PBMC) were isolated from blood cones obtained from healthy donors (NHS Blood and Transplant [NHSBT] UK) by LSM 1077 (PAA) gradient centrifugation and then incubated with CD25+ Microbeads to derive CD25+ enriched cells using a LS column (Miltenyi Biotech). CD25+ cells were stained with monoclonal antibodies and Treg cell subsets were isolated by FACS using a BD FACSAria I cell sorter.
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study
| 99.94 |
The total CD4+CD127−/lowCD25+ Treg cell (total Treg cells) population (purity: mean, 91.9% (82.8-95.9%) of total sorted live CD4+ cells) was sorted into 3 phenotypically distinct subpopulations, based on expression of CD25 and the naive T cell marker CD45RA: CD127−/lowCD25intCD45RA− (CD25int memory Treg [mTreg]) and CD127−/lowCD25hiCD45RA− memory Treg (CD25hi mTreg); and CD127−/lowCD25intCD45RA+ naive Treg (naive Treg) (Figure 1A). Purity of sorted populations was mean 87.6% (81.0-95.3%) CD25int mTreg, mean 73.8% (63.9-84.7%) CD25hi mTreg and mean 75.6% (36.9-94.9%) naive Treg of total live CD4+ cells.
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study
| 100.0 |
Treg phenotyping. A, Gating strategies to isolate total Treg (CD127−/lowCD25+); naive Treg (CD127−/lowCD25intCD45RA+); CD25int mTreg (CD127−/lowCD25intCD45RA−); and CD25hi mTreg (CD127−/lowCD25hiCD45RA−). B, Percentages of cells with demethylated TSDR (n = 11) and (C) percentage of cells that express FOXP3 (n = 12) before expansion (Mann-Whitney U test, *P < 0.05, **P < 0.01, ***P < 0.001). Median with interquartile range is represented.
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study
| 100.0 |
Expansion protocol was adapted from previous protocols with minor modifications.10 Sorted cells were expanded in Roswell Park Memorial Institute 1640 media supplemented with l-glutamine, penicillin/streptomycin, sodium pyruvate, and 10% of human AB pooled serum, in the presence of recombinant human IL-2 (1000 U/mL) (Novartis) and Dynabead Human T-activator CD3/CD28 (Life Technologies) in a 1:1 cell to bead ratio over two 7-day rounds of expansion. During the second round of expansion, 8 ng/mL TAC (Sigma) dissolved in dimethyl sulfoxide (DMSO) (Sigma), based on the published trough level in patients with stable liver or kidney allografts,6,15,16 or empty DMSO control added to cell media (final concentration of DMSO in culture was 1.92 × 10−4% volume).
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study
| 100.0 |
Expanded Treg suppressive capacity was assessed in 72 hours in vitro assays in the absence of TAC. Variable numbers of Treg were incubated with 10 × 104 autologous PBMC labelled with 1 μM VPD450 (BD) proliferation dye in triplicate wells containing 2 × 104 αCD3/αCD28 beads in 96-well plates. Labeled PBMC cultured alone in the presence of αCD3/αCD28 beads were used as a negative control for suppression. After a 72-hour incubation, cocultures were stained to distinguish CD4+ and CD8+ cells, and violet proliferation dye dilution was analyzed by flow cytometry. The percentage of PBMC suppression was calculated by using division index of cocultures containing Treg and PBMC compared with division index of PBMC alone, according to the following formula (1 − (div.index Treg + PBMC/div.index PBMC)). Data were analyzed with FlowJo software version 9.5.3. The program calculates division index as the average of cell divisions that a cell in the original population has undergone.
|
study
| 100.0 |
Regulatory T cell–specific demethylation region DNA methylation analysis was performed as previously described17 using genomic DNA isolated from freshly sorted or expanded Treg cells using the QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany). A minimum of 60-ng bisulfite-treated (EpiTect; Qiagen) genomic DNA was used in a real-time polymerase chain reaction (PCR) to quantify the Foxp3 TSDR. Real-time PCR was performed in a final reaction volume of 20 μL containing 10-μL FastStart universal probe master (Roche Diagnostics, Mannheim, Germany), 50-ng/μL lamda DNA (New England Biolabs, Frankfurt, Germany), 5-pmol/μL methylation or nonmethylation-specific probe, 30-pmol/μL methylation or nonmethylation-specific primers, and 60-ng bisulfite-treated DNA or a respective amount of plasmid standard. The samples were analyzed in triplicates on an ABI 7500 cycler and reported as % T cells with demethylated TSDR region.
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study
| 100.0 |
Two-week expanded Treg cells were thawed and expanded further for 2 days in DMSO or TAC containing media with IL-2 (1000 U/mL) and with 2 × 104 αCD3/αCD28 beads (1:5 bead:Treg) or without bead stimulation as a negative control. Supernatants were then centrifuged and stored at −80°C until analyzed. IL-10, IL-17A, and IFN-γ levels were determined using enzyme-linked immunosorbent assay according to manufacturers' protocol (BD for IL-10 or eBiosciences for IL-17A and IFN-γ).
|
study
| 100.0 |
Preliminary analysis demonstrated a large variation in phenotype, suppressive function, expansion, and susceptibility to TAC-induced alteration of Treg function between CD4+CD25+CD127−/lo cells (total Treg cells) isolated from different healthy human donors (data not shown). It is possible that heterogeneity in total CD4+CD25+CD127−/lo Treg cells between donors accounted for these variations. Further experiments were therefore performed by costaining with CD45RA in an attempt to define a more homogeneous subpopulation of Treg present consistently in different donors. Total Treg cells were sorted into naive Treg cell, CD25int mTreg cells, and CD25hi mTreg cells as described in the Methods (Figure 1A).
|
study
| 100.0 |
Approximately 91% of total Treg had a demethylated TSDR before expansion (median, 91.1%). A significantly larger proportion of the CD25hi mTreg subpopulation showed a demethylated TSDR (median, 96.2%) compared with naive Treg (median, 83.5%) and CD25int mTreg (median, 84.6%) (Figure 1B).
|
study
| 100.0 |
A high proportion of cells expressing FOXP3 protein corresponded to a high proportion of cells with a demethylated TSDR in sorted populations (Figures 1B and C., respectively). Similar to TSDR demethylation results, the proportion of cells expressing Foxp3 tended to be higher in CD25hi mTreg (median, 93.6%) compared with CD25int mTreg (median, 83.7%), naive Treg (median, 78.7%), and total Treg (median, 85.2%) (Figure 1C).
|
study
| 100.0 |
It is possible that successful induction of immunological unresponsiveness after transfusion of Treg cells into patients will require further expansion in vivo to both control allospecific immunity adequately and replace Treg cells that die as part of natural cell turnover. In this circumstance, Treg cells will need to expand in vivo despite exposure to pharmacological immunosuppression.
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other
| 99.9 |
To investigate whether TAC differently affected the expansion of Treg subpopulations, we analyzed the effect of TAC on cell expansion in the final 7 days of a 14-day culture, comparing cell numbers at day 7 with day 14, when cells were exposed to TAC (8 ng/mL) or DMSO vehicle control. CD25hi mTreg were highly anergic and could not be expanded sufficiently to perform experiments. As expected, TAC significantly reduced expansion of all Treg populations. CD25int mTreg expanded a median of 25- and 13-fold; naive Treg by a median of 21- and 6-fold; and total Treg cells expanded a median of 16- and 6-fold in the presence of DMSO or TAC, respectively (Figure 2A). Analysis of the ratio of proliferation between Treg incubated with DMSO and TAC demonstrated that TAC did not inhibit proliferation of any 1 Treg cell subpopulation more than any other (Figure 2B).
|
study
| 100.0 |
Expansion capacity of Treg cell populations. A, Fold expansion in each Treg cell population was measured after in vitro expansion in the presence of DMSO or TAC (Wilcoxon-matched pair test), comparing day 7 with day 14 cell numbers. B, Ratio of proliferation between each Treg cell population incubated with DMSO or TAC (Mann-Whitney U test). n = 12 donors are shown (*P < 0.05, **P < 0.01, ***P < 0.001). Median with interquartile range is represented.
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study
| 100.0 |
Next, the stability of the phenotype of total Treg cells, naive Treg cells, and CD25int mTreg cells after expansion without TAC was examined. FOXP3 expression and TSDR demethylation were measured on day 0 and day 14, suppressive function was determined on day 14.
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study
| 100.0 |
Before expansion, the majority of naive Treg and CD25int mTreg expressed FOXP3 and possessed a demethylated TSDR (Figure 1). Naive Treg and CD25int mTreg cells showed differential stability of this Treg cell phenotype during expansion in DMSO control media. A significantly higher proportion of CD25int mTreg cells did not have a demethylated TSDR phenotype (median, 14.2% remaining demethylated) compared to the naive Treg cell subpopulation (median, 78.7% remaining demethylated) (Figure 3A, P < 0.05) by day 14. This finding correlated with a substantial reduction in the population that expressed CD27, a marker known to be associated with highly suppressive Treg,18 by CD25int mTreg compared with naive Treg expanded in DMSO control media (Figure 3C).
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study
| 100.0 |
Foxp3 expression and demethylation status of the TSDR region of expanded Treg cell subsets. A, Percentages of TSDR demethylation (n = 8 donors); B, cells expressing Foxp3 (n = 8 donors); C, cells expressing CD27 (n = 7 donors); and D, ratio of cells that express FOXP3 and with a demethylated TSDR were measured in each Treg cell population before and after in vitro expansion in the presence of DMSO or TAC. For statistical analysis, Wilcoxon-matched pair test was performed comparing, CD27 expression, Foxp3 expression, TSDR demethylation or FOXP3+/demethylated TSDR ratio between the respective Treg population after being exposing to DMSO or TAC, and the Mann-Whitney U test was used to compare between cell populations (*P < 0.05, **P < 0.01, ***P < 0.001). Median with interquartile range is represented.
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study
| 100.0 |
CD25int mTreg maintained similar FOXP3 protein expression before and after expansion (median, 83.7% and 74.9%, respectively) in DMSO control media, whereas the proportion of naive Treg that expressed FOXP3 increased (median, 78.7% to 95.8% after expansion) (Figure 3B). As the extensive loss of TSDR demethylation status in the CD25int mTreg population is not reflected by reduced FOXP3 expression, it appears that the CD25int expanded population contains a greater proportion of activated Teff cell or induced Treg (iTreg) cell that may transiently express FOXP3 without having a demethylated TSDR. A reduced level of TSDR demethylation alongside a sustained FOXP3 expression altered the ratio of FOXP3 expressing cells compared with TSDR demethylated cells after expansion in the CD25int mTreg population (Figure 3D).
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study
| 100.0 |
When comparing preexpanded and cells expanded in DMSO, total Treg cells showed a level of TSDR demethylation (median, 25.2% retain) and Foxp3 expression (median, 87.4% retain) that was intermediate to naive Treg and CD25int mTreg subpopulations (data not shown) to manifest an enhanced ratio of FOXP3 expression to TSDR demethylation (Figure 3D), that is probably due to inclusion of CD25int mTreg when sorted on day 0.
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study
| 100.0 |
Despite presenting higher levels of TSDR demethylation and Foxp3 expression, expanded naive Treg cells were not significantly more suppressive than CD25int mTreg cells (Figure 4A); suppression was similar between total Treg cells, CD25int mTreg cells, and naive Tres cells after expansion in DMSO control media (Figure 4A). This indicates that those CD25int mTreg cells that express FOXP3 but do not retain a demethylated TSDR are iTreg cells that are capable of suppressing.
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study
| 100.0 |
In vitro suppressive capacity of Treg cell populations. A, Suppressive capacity of CD25int mTreg and naive Treg (n = 6 donors), and total Treg (n = 5 donors) after in vitro expansion in DMSO control media. B, Suppressive capacity of CD25int mTreg and naive Treg (n = 6 and n = 5 donors, respectively), and total Treg cells (n = 5 donors) after in vitro expansion in the presence of TAC. Percent suppression of CD4+ effector cell proliferation based on division index of PBMCs compared with the proliferation of PBMCs cultured in the absence of suppressor cells. Each data point is the average of 3 replicate wells in the suppression assay of each donor. Mann-Whitney U test used to compare suppression between Treg subpopulations (*P < 0.05). Mean with SEM is represented. C, Ratio of percentage suppression between Treg expanded in DMSO and TAC containing media, using titrated numbers of Treg to Teff cells. (n = 6 donors; Mann-Whitney U test, *P < 0.05). Median with interquartile range is represented.
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study
| 100.0 |
Tacrolimus has been shown to downregulate FOXP3 expression, whereas TSDR demethylation remained stable in allograft recipient patients.6,19 In keeping with these findings, we observed that the percentage of CD25int mTreg and naive Treg cells (Figure 3A) with demethylated TSDR was not different between cells expanded in TAC or DMSO control containing media. In contrast, cells expanded with TAC showed reduced FOXP3 expression in naive Treg and CD25int mTreg cell populations (P < 0.01, P < 0.05, respectively; Figure 3B).
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study
| 100.0 |
Total Treg, CD25int mTreg, and naive Treg showed equal suppressive function after expansion in DMSO (Figure 4A); however, suppressive function of naive Treg expanded in TAC appeared to be partially abrogated compared with CD25int mTreg, when lower ratios of Treg-Teff were examined (Figure 4B; 1:16 ratio P < 0.05). When analyzing the suppressive capacity of Treg generated from 6 donors independently, a clear trend was observed that TAC reduced suppression of the naive Treg subpopulation in a larger proportion of donors compared with the CD25int mTreg cell subpopulation (2-sided Fisher exact test: P = 0.08). Tacrolimus impaired suppressive capacity of CD25int mTreg in only 1 of 6 donors (Figure 1A, SDC, http://links.lww.com/TP/B286); in contrast, in 4 of 5 donors, naive Treg showed reduced suppressive activity after expansion with TAC (sufficient numbers of naive Treg expanded with TAC could not be obtained from 1/6 donors) (Figure 1B, SDC, http://links.lww.com/TP/B286). An examination of the ratio of suppression by Treg expanded in DMSO and TAC showed that the naive Treg population was more susceptible to TAC mediated reduction of suppression than the CD25int mTreg population (Figure 4C).
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study
| 100.0 |
The greater stability of the TSDR demethylation status and CD27 expression by naive Treg cells compared with CD25int mTreg cell during expansion showed important differences between these populations. Critically, an enhanced stability of suppressive function after expansion in TAC by CD25int mTreg cells compared with naive Treg cells prompted the measurement of the suppressive cytokine IL-10 and any divergence in its production between Treg cell populations.
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study
| 100.0 |
IL-10 production was accessed in supernatant after expansion of CD25int mTreg and naive Treg cells in TAC or DMSO in 2 donors (Figure 5A, donor 1; B, donor 2). CD25int mTreg produced substantially more IL-10 than naive Treg cells. Expansion when exposed to TAC significantly reduced IL-10 production by both Treg cell populations, with IL-10 production by naive Treg cell almost completely ablated but production by CD25int mTreg remaining relatively high. Remaining IL-10 production when exposed to TAC by CD25int mTreg cells may explain the greater stability of suppressive function of CD25int mTreg cells compared with naive Treg cells.
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study
| 100.0 |
Cytokine production of Treg cell subpopulations. IL-10, IFN-γ, and IL-17A production by CD25int mTreg and naive Treg cells after in vitro expansion in the presence of TAC or DMSO was determined in triplicate wells in 2 different donors (A and B). Bars represent means with SEM (***P < 0.001).
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study
| 100.0 |
Given the reduced percentage of CD25int mTreg that possess a demethylated TSDR after expansion compared with naive Treg cells (Figure 3A), it is possible that CD25int mTreg may have the potential to revert to an effector function. To test whether the reduced TSDR demethylation corresponds with enhanced effector function, we examined IFN-γ and IL-17A production after expansion. Indeed, CD25int mTreg produce substantially greater levels of both IFN-γ and IL-17A compared with naive Treg (Figure 5). Tacrolimus significantly reduced both IL-17A and IFN-γ production (Figure 5).
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study
| 100.0 |
Both CD25int mTreg and naive Treg cell subpopulations had a similar high percentage of cells with a demethylated TSDR and FOXP3 expression before expansion. Loss of TSDR demethylation during expansion was restricted to CD25int mTreg cells. It is possible that the CD25int mTreg cell population is contaminated with memory Teff cell that expand faster than CD25int mTreg cell to become the predominant constituent of cultured CD127−/loCD25intCD45RA− sorted cells after 14 days of polyclonal stimulation. Regulatory T cell TSDR demethylation status is described to be highly stable when cells are subjected to extended periods of in vitro expansion.12 It is therefore less likely that CD127−/loCD25+CD45RA− cells that are TSDR demethylated on day 0 undergo epigenetic alteration to possess a methylated TSDR during expansion. Both CD25int mTreg and naive Treg cells express high levels of FOXP3 after expansion, yet the former may be accounted for by CD25int Teff cell contaminants expressing FOXP3 after activation. The combined observations of a greater level of IL-10 production, a lack of a TSDR demethylated status, and expression of FOXP3 by CD25int mTreg expanded cells strongly indicates that a large proportion of this population is comprised of de novo produced iTreg cells that may derive from Teff cells that contaminate the sorted population of day 0 and expand to form the main population constituent. Contaminating CD4+ Teff cells may have been converted to iTreg cells in TGF-β–rich culture conditions,20 derived from conventional Treg cells21 sorted alongside. The contaminating non-Treg population may have comprised a small constituent of the sorted CD25int mTreg cell population on day 0 because the proportion of CD25int mTreg and naive Treg with demethylated TSDR is similar at day 0. The dominant method by which naive Treg and CD25int mTreg exert suppression may differ, given the differential level of IL-10 production and susceptibility of interference of suppression by TAC.
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study
| 100.0 |
The large drop in the proportion of CD25int mTreg with a demethylated TSDR post expansion, regardless of the exposure to TAC, identifies a considerable risk to adoptively transferring these cells into patients to tolerize allograft recipients. Although the CD25int mTreg population showed superior IL-10 production and stability to suppress when expanded in the presence of TAC, the lack of a demethylated TSDR is known to identify cells with an unstable Treg phenotype that are susceptible to loosing suppressive function.22 Indeed, we observed that CD25int mTreg produce substantially greater amounts of IL-17A and IFN-γ compared with naive Treg. Regulatory T cells with an unstable regulatory phenotype have been shown to have the potential to develop into pathogenic cells in mice23 and similar could be true for human Treg cells. It is possible that CD25int mTreg cells, possibly containing a large proportion of cells derived from Teff cell contaminants on day 0 that expand faster than conventional Treg, will lose suppressive function after adoptive transfer to patients and damage the allograft.
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study
| 100.0 |
We show that a naive Treg population may be sorted using surface markers and expanded to comprise a promising option for future studies to adoptively transfer Treg cells to tolerize an allograft. Despite some loss of Treg function in the presence of TAC, this population of cells nevertheless retains a high degree of suppressive function and importantly retains a TSDR demethylated status after expansion. This study indicates that sorting a population of naive Treg cells according to a CD4+CD45RA+CD25intCD127−/lo will improve therapy beyond sorting the whole CD4+CD25+CD127lo total Treg cell population. Future work may identify if sorting a mixture of naive Treg and CD25hi mTreg will further enhance the production of a suitable Treg population for cellular therapy. Analysis of suppressive function in a freshly isolated nonexpanded total Treg cell population in patients undergoing haemodialysis showed a reduced suppressive function compared with healthy controls.24 Future studies may establish if the naive Treg population that we examined here has reduced capacity to suppress compared with the same population obtained from healthy subjects.
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study
| 100.0 |
Agricultural producers are facing increasing pressure to reduce the use of fungicides, for which deployment of cultivars with disease resistance is one viable solution. Accordingly, several sources of resistance to major crop diseases have been identified and introgressed. For example, in grapes the two most important foliar diseases, powdery mildew and downy mildew, can be suppressed by resistance genes identified from wild sources (Blasi et al. 2011; Feechan et al. 2013; Mahanil et al. 2012; Ramming et al. 2011). Once genetic control of major diseases and the subsequent reduction in fungicide applications is achieved, other problems may emerge, namely pathogens that were secondary targets of routine fungicide applications. This phenomenon has been observed in powdery and downy mildew-resistant vineyards, where the incidence of grapevine black rot increased (Molitor and Beyer 2014; Rex et al. 2014).
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study
| 99.44 |
Phomopsis cane and leaf spot of grapevine (“Phomopsis”) is caused by Diaporthe ampelina (Ascomycota, Diaporthales; syn. = Phomopsis viticola,) (Gomes et al. 2013; Wilcox et al. 2015). Most Diaporthe species are considered hemibiotrophic (Udayanga et al. 2011), with an initial biotrophic phase of plant tissue colonization before the necrotrophic phase, when lesions or cankers develop. In grapevine, leaf and cane infections by D. ampelina are initiated by rain-splashed conidia released from pycnidia present on previously infected tissues. Dispersed conidia adhere to the plant tissues and under suitable conditions, germinate and penetrate tissues through stomatal pores or wounds (Pine 1959). Leaf and cane infections require a minimum of 7 h wetness duration at optimum temperatures of 16–20 °C (Erincik et al. 2003). In plant tissues, the mycelium germinating from conidia invades the cortical parenchyma and forms pseudo-parenchymatous mats among host cells. Host cells become necrotic and shoot lesions and leaf spots usually appear 3–4 weeks after infection (Wilcox et al. 2015). New pycnidia form on these necrotic lesions, providing inoculum for new infections. Lesions remain after lignification in dormant canes, resulting in shoot breakage. On clusters, Phomopsis can cause lesions on the rachis, resulting in loss of up to 30% of yields (Anco et al. 2012).
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study
| 99.94 |
In Mediterranean climates (e.g., California), foliar symptoms are less common, but D. ampelina and other Diaporthe species are instead more frequently associated with the formation of wood cankers (Lawrence et al. 2015), Phomopsis dieback being part of the grapevine trunk-disease complex (Úrbez-Torres et al. 2013). In controlled experiments, grapevine cultivars responded differently to wood infection by D. ampelina (Travadon et al. 2013), suggesting a genetic component in the plant–pathogen interaction. To date, the genetic and molecular bases of Phomopsis resistance in grapevines have not been reported.
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study
| 99.94 |
In the plant immune response, pathogen-associated molecular patterns (PAMPs) are recognized by pathogen recognition receptors, triggering a defense response known as PAMP-triggered immunity (PTI). The pathogen can escape this defense response by deploying effectors. In response, plants utilize a surveillance mechanism mediated by R-genes coding for proteins characterized by a nucleotide-binding site leucine-rich repeats (NB-LRR). Upon recognition of pathogen effectors, a cascade of reactions leads to a hypersensitive response [effector-triggered immunity (ETI)] (Jones and Dangl 2006). This type of response is associated with the production of reactive oxygen molecules and localized cell death, mediating the resistance to biotrophic and hemibiotrophic fungi (Greenberg and Yao 2004; Morel and Dangl 1997). Defenses against biotrophic pathogens are also regulated by a salicylic acid (SA)-dependent pathway, which plays a role in both local defense reactions and induction of systemic acquired resistance (Durner et al. 1997). In contrast, defenses against necrotrophic pathogens are regulated by induction of jasmonic acid (JA) and ethylene signaling (Glazebrook 2005). In the plant defense response, there is an antagonistic cross talk between SA and both ethylene and JA pathways, as well as SA and auxin signaling pathways (Kazan and Manners 2009).
|
review
| 99.6 |
R-genes are often major dominant genes that provide complete or qualitative disease resistance, becoming interesting targets for introgression in breeding programs. Over time, plant pathogens can modify their effectors, avoiding recognition, and thus resistance mediated by R-genes can be overcome in new cultivars after their deployment (Jones and Dangl 2006; Peressotti et al. 2010). Stacking of several loci has been proposed as a mechanism to prolong the durability of R-genes, but the selection of multiple loci that generate the same phenotype requires the use of molecular markers through marker-assisted selection (MAS).
|
review
| 99.1 |
In this paper, we report our study into the genetics of Phomopsis resistance of canes and clusters in three hybrid grapevine families. First, we quantified the segregation of cane and cluster symptoms in families derived from interspecific hybrids ‘Horizon’ and Illinois 547-1, V. vinifera ‘Chardonnay’, and V. cinerea B9. We used high-density genetic maps to study the association between phenotype and molecular markers, which allowed further identification of two novel major resistance loci. Candidate genes were further dissected through gene expression analysis.
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study
| 100.0 |
Three related full sib, F1 families were derived from the cross of four parental genotypes: V. vinifera ‘Chardonnay’ clone 95, V. cinerea B9, Illinois 547-1 (V. rupestris B38 × V. cinerea B9) and the complex hybrid ‘Horizon’ (‘Seyval’ × ‘Schuyler’, whose pedigree includes V. vinifera, V. labrusca, V. aestivalis and V. rupestris (Reisch et al. 1982). The ‘Horizon’ × Illinois 547-1 family (366 vines) resulted from crosses made in 1988 (Dalbó et al. 2000) and 1996. Families ‘Horizon’ × V. cinerea B9 (162 vines) and ‘Chardonnay’ × V. cinerea B9 (148 vines) resulted from crosses made in 2009. For all families, in the year following cross-hybridization, seeds were stratified prior to germination, and seedlings were grown in an irrigated field nursery. Two years after cross-hybridization, vines were transplanted to a permanent vineyard in Geneva, New York. Single vines per genotype were planted 1.2 m apart. A control block was included in each row, with the following genotypes: V. vinifera ‘Chardonnay’ (susceptible to powdery and downy mildew), V. hybrid ‘Chancellor’ (Seibel 5163 × Seibel 880) (susceptible to powdery and downy mildew), V. rupestris B38 (resistant to powdery and downy mildew, V. hybrid ‘Horizon’ (‘Seyval’ × ‘Schuyler’) (intermediate resistance to powdery and downy mildew) and the breeding selection NY88.0514.04 (resistant to powdery and downy mildew). Powdery mildew-susceptible control ‘Chardonnay’ was planted after every 15 seedling vines. Parental lines V. cinerea B9 and Ill. 547-1 are also classified as resistant to powdery and downy mildew.
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study
| 99.94 |
Fungicide applications were reduced to the minimum necessary to maintain plant viability. N-trichloromethylthio-4-cyclohexene-1,2-dicarboximide (Captan 80WDG) was applied at recommended rates at the following phenological stages [identified according to the modified Eichhorn–Lorenz scale (Coombe 1995)] during 2011 through 2013: stage 12 (1.68 kg/ha, late May), stage 17–18 (2.24 kg/ha, early June), stage 26 (2.80 kg/ha, mid-June), stage 29 (2.80 kg/ha, late June) and stage 31 (2.80 kg/ha, mid-July). Potassium phosphite (ProPhyt, Helena Chemical Company, Collierville, TN, USA) was applied for control of downy mildew at a rate of 2.35 kg/ha of phosphorous acid equivalent at stages 32 and 34 (early and mid-August, respectively) in 2011 and 2012.
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other
| 82.8 |
After assessment of symptoms, field vines were vegetatively propagated for further experiments. First, V. cinerea B9 vines were propagated in 2010 as described previously (Barba et al. 2015): briefly, dormant cuttings were taken from the vineyard, stored at 4 °C and potted in a 3:1 mixture of perlite: soil with bottom heat at 26 °C until sufficient rooting took place. Vines were then grown in a greenhouse under a 16 h photoperiod at 27–30 °C, pruned and stored at 4 °C for dormancy. In 2011, potted vines were grown in a greenhouse as described above and pruned to assure uniform vegetative growth when needed. Secondly, dormant cuttings from resistant and susceptible plants were sent to California to confirm phenotypes under controlled conditions (i.e., symptoms were due to D. ampelina alone). From cross ‘Chardonnay’ × V. cinerea B9, four resistant progenies (454064, 455035, 454053, 454058) and six susceptible progenies (454066, 454071, 455072, 455082, 454045, 454077) were propagated. Twelve replicates per genotype were established in the greenhouse at the University of California Experiment Station in Davis as a source of green cuttings for the following experiments. Dormant cuttings taken from the New York vineyard were surface sterilized in 1% sodium hypochlorite for 15 min, soaked in water overnight and then callused in boxes filled with perlite and vermiculite (1:1, vol/vol) for 21 days at 30 °C and 85% relative humidity. After callusing, cuttings were planted in sleeves in a 1:1 mixture of perlite and vermiculite and then returned to 30 °C at 85% relative humidity for 14 days to further encourage callusing. Plants were afterward placed under intermittent water mist (5 s every 2 min during daylight) at 28 °C for 7 days in the greenhouse, at which point leaves emerged and plants were then transferred to the greenhouse and potted in UC mix (Baker 1957). After 5 months [natural sunlight photoperiod, 25 ± 1 °C (day) and 18 ± 3 °C (night)], there was sufficient shoot growth for propagation of plants from green cuttings for inoculation experiments.
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study
| 100.0 |
Field vines were subject to natural inoculation. Phomopsis symptoms were evaluated on dormant canes each autumn from 2011 through 2013 using the following disease severity scale: (0) no symptoms; (1) light infection, few discrete circular lesions; (2) moderate infection, widespread coalescing circular lesions; (3) severe infection, widespread coalescing misshapen lesions with blackened surface and corky texture (Fig. 1a). Symptoms on clusters were scored as present or absent before veraison in 2013 and 2014. As male vines did not set fruit, the number of samples was reduced to 65 observations in the ‘Horizon’ × V. cinerea B9 family and 58 observations in the ‘Chardonnay’ × V. cinerea B9 family. No cluster observations were made in the ‘Horizon’ × Illinois 547-1 family.Fig. 1Symptoms and Diaporthe ampelina isolation. a Phomopsis cane symptoms were scored on dormant canes using the following scale: (0) no Phomopsis symptoms observed; (1) light infection, small number of discrete lesions; (2) moderate infection, lesions coalescing, widespread; and (3) severe infection, lesions blackened, internode tissue corky and misshapen. b D. ampelina culture isolated from symptomatic canes (score 3), growing on potato dextrose agar. c Progression of symptoms on resistant (left) and susceptible (right) full siblings growing side by side in the vineyard on August 21 (upper) and September 10 (lower), 2013. d Phomopsis symptoms on green shoots and unripe berries
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study
| 100.0 |
Symptoms and Diaporthe ampelina isolation. a Phomopsis cane symptoms were scored on dormant canes using the following scale: (0) no Phomopsis symptoms observed; (1) light infection, small number of discrete lesions; (2) moderate infection, lesions coalescing, widespread; and (3) severe infection, lesions blackened, internode tissue corky and misshapen. b D. ampelina culture isolated from symptomatic canes (score 3), growing on potato dextrose agar. c Progression of symptoms on resistant (left) and susceptible (right) full siblings growing side by side in the vineyard on August 21 (upper) and September 10 (lower), 2013. d Phomopsis symptoms on green shoots and unripe berries
|
study
| 99.9 |
Canes from diseased vines located in Geneva, NY, were collected during the spring of 2013 and incubated in a clean, sealed plastic box with wet paper towels to provide humidity. Diaporthe ampelina conidia were collected from oozing lesions and plated on potato dextrose agar (PDA, Difco Laboratories, Detroit, MI, USA), and emerging colonies were subcultured onto fresh PDA plates. Cultures were maintained at room temperature under fluorescent light and transferred to fresh PDA every 3–4 weeks. For RNA-Seq experiments, controlled inoculations were made using a solution of conidia obtained by flooding pycnidia-bearing colonies on PDA plates with sterile distilled water; after approximately 5 min, the resulting spore suspension was decanted and diluted in sterile water plus Tween 20 (10 µl/l) to a final concentration of 107 conidia/ml.
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study
| 100.0 |
To confirm the phenotypes observed under NY field conditions were due to D. ampelina alone, the following experiment was performed: Two-bud green cuttings were taken from green shoots of California greenhouse stock plants in October 2015, December 2015 and February 2016, corresponding to three independent, replicated experiments. Cuttings were rooted in perlite in the greenhouse [natural sunlight photoperiod, 25 ± 1 °C (day), 18 ± 3 °C (night)] with intermittent water mist (5 s every 2 min during daylight). Roots formed after 2 weeks, at which point cuttings were transplanted into a potting mix of peat, sand and perlite (1:1:1, v/v/v) in plastic trays (58 × 40 × 22.5 cm; XL High Dome Propagator, Garland Products, England). For each of the three experiments, a total of 24 inoculated plants per genotype [parental vines V. cinerea B9 and ‘Chardonnay’, four resistant progenies (454064, 455035, 454053, 454058) and six susceptible progenies (454066, 454071, 455072, 455082, 454045, 454077)] were evenly divided among four trays (six plants per genotype per tray), with replicate plants planted in a row. A separated tray with six plants per genotype was prepared for non-inoculated control. After 4 weeks, at least five leaves were present on each plant and the youngest internode was tagged.
|
study
| 100.0 |
Spores from D. ampelina isolate Nita001 were produced according to Travadon et al. (2013) from an isolate originally collected in June 2015 from leaf spots on ‘Cabernet Sauvignon’ in Winchester, Virginia. The spore suspension was adjusted with sterile water to 1 × 106 conidia ml−1 and was sprayed until runoff on the leaves and stems of plants using an atomizer (Mondi Mist & Spray Deluxe Tank Sprayer, Hydroframs, USA). Non-inoculated plants were sprayed in the same way, but with sterile water instead. Infection was encouraged by covering each tray with a dome (XL High Dome Propagator, Garland Products, England), maintaining greenhouse conditions at 20 °C and continuous light for 24 h. After this 24-h wetting period, domes were removed from each tray.
|
study
| 100.0 |
Disease severity on the internodes was assessed 30 days post-inoculation by estimating the percentage of the area covered by lesions on the four internodes below the internode tagged at inoculation, using a modified Horsfall–Barratt scale (Barratt and Horsfall 1945) with 12 levels (1: 0–5%; 2: 5–10%; 3: 10–20%; 4: 20–30%; 5: 30–40%; 6: 40–50%; 7: 50–60%; 8: 60–70%; 9: 70–80%; 10: 80–90%; 11: 90–100%; 12: 100%). Differences between genotypes were determined by ANOVA, as described in “Statistics”.
|
study
| 100.0 |
Six 1-year-old, 1 m tall V. cinerea B9 plants were acclimated in a lighted mist chamber (25 °C) 2 days before inoculation. Leaves were spray inoculated with a Preval handheld paint sprayer (Preval, IL, USA) using either a D. ampelina suspension isolated from field-infected vines as described, or sterile water (mock).
|
study
| 99.94 |
One leaf sample for each inoculation treatment was collected from three replicate vines (biological replicates) both before (T0, 3rd leaf) and 48 h post-inoculation (hpi) (T2, 4th leaf) (three replicates for each of two collection times for each of two inoculations conditions = 12 samples). Tissues were immediately stored in liquid nitrogen and transferred to the laboratory for RNA extraction. Total RNA was extracted using a Spectrum Plant Total RNA kit (Sigma-Aldrich, USA), after grinding frozen tissue to a fine powder with mortar and pestle. Barcoded, strand-specific, mRNA multiplexed libraries were prepared as previously described (Zhong et al. 2011). Each library was single-end (100 bp) sequenced on a HiSeq 2000 (Illumina Inc., USA) at the Genomics Facility of the Institute of Biotechnology at Cornell University.
|
study
| 100.0 |
RNA-Seq reads were processed with the Fastx toolkit for demultiplexing, barcode trimming and quality filtering (Pearson et al. 1997). Cutadapt was used to remove all residual adapter sequences (Martin 2011). Differential expression analysis of normalized FPKM (fragments per kilobase of exon per million fragments mapped) expression values was executed following standard protocols (Haas et al. 2013), with the following experiment-specific details. First, the RSEM software (Li and Dewey 2011) was used to align the quality reads to the V. vinifera PN40024 reference transcriptome (Grimplet et al. 2012). The trimmed mean of M-values (TMM) normalization method was executed in R to generate FPKM values for each transcript (Dillies et al. 2013).
|
study
| 100.0 |
After calculation of normalized expression values for each sample, DE genes after inoculation [false-discovery rate (FDR) ≤ 0.001] were determined for each inoculation treatment using the edgeR software (Robinson et al. 2010). The set of exclusive DE genes in samples inoculated with D. ampelina was obtained by subtracting genes that were DE after both pathogen and mock inoculation. These inoculated-exclusive DE genes were input for pathway enrichment analysis as previously described (Osier 2016). The experiment described in the above section is referred to as a DE study in the following sections.
|
study
| 100.0 |
Genotyping and genetic map construction for these families have been previously described (Hyma et al. 2015). Briefly, DNA was extracted from one young leaf per vine using the DNeasy® 96 Plant Kit (Qiagen). Genotyping-by-sequencing (GBS) libraries (Elshire et al. 2011) were constructed at 384-plex and sequenced with an Illumina HiSeq 2000 DNA sequencer (single-end, 100 bp read length). SNP calling was performed according to the TASSEL 3.0 GBS pipeline (Glaubitz et al. 2014) using the V. vinifera PN40024 reference genome version 12X.0 (Adam-Blondon et al. 2011; Jaillon et al. 2007). SNP names indicate SNP position on the reference genome coded as S(chromosome)_(position in bp). GBS genotype information was used to identify vines derived from self-pollination or cross-contamination, which were removed from the family dataset. SNP filtering and parental genetic map construction (Table 1) utilized the de novo HetMappS pipeline, using pseudo-testcross markers only (Hyma et al. 2015).Table 1Total genetic map distance and number of SNPs for female and male maps of three Vitis F1 familiesFamily (# individuals)Genetic distance (cM)Number of SNPsFemale mapMale mapFemale mapMale map‘Horizon’ × Illinois 547-1 (366)1286131443165560‘Horizon’ × V. cinerea B9 (162)1347112531181956‘Chardonnay’ × V. cinerea B9 (148)1275129323942177Genetic maps were created using the HetMappS de novo pipeline and curated with R/qtl
|
study
| 100.0 |
Additionally, for a subset of 94 DNA samples of progeny and parents of the ‘Horizon’ × V. cinerea B9 family, the following SSR markers located near the resistance loci were genotyped: VVIB22 (Merdinoglu et al. 2005), VrZAG62 (Sefc et al. 1999), VVMD7 (Bowers et al. 1996) and SC8_0040_088 (Jaillon et al. 2007). PCR reactions were performed with 6 µl of QIAGEN Multiplex PCR Plus Kit (Qiagen, Germany), 1 µl of primer mix (0.5 µM each) and 5 µl of each DNA sample diluted 1:10. PCR amplification was performed with 30 cycles of 95 °C for 30 s, 57 °C for 90 s and 72 °C for 90 s, followed by 68 °C for 30 min. Fragment sizes were determined relative to a LIZ 500 Size Standard using an ABI 3730xl (Applied Biosystems, USA) at the Genomics Facility of the Institute of Biotechnology at Cornell University. Allele calls were generated using GeneMarker V 2.4.0 (SoftGenetics, USA).
|
study
| 100.0 |
QTL were localized using the R/qtl package (Broman et al. 2003) implemented in the statistical software R (R Core Team 2014) as described previously (Barba et al. 2014). Multipoint probabilities were calculated using calc.genoprob with step = 1 and default parameters. Initial QTL positions were determined with the scanone function using a normal model, Haley–Knott regression and default parameters. LOD significance scores were determined by permutation tests (1000). Initial QTL positions were used to define QTL with the makeqtl function; significance of model terms was tested with fitqtl command and positions were refined with refineqtl. The addqtl command was used to test if another QTL was needed. A 1.5 LOD supported interval was determined using the lodint function, and QTL effects were calculated as the difference in the mean phenotype value of individuals within each genotype class at the marker or pseudomarker (a position between markers) with the highest LOD score, using the effectplot function in R/qtl.
|
study
| 100.0 |
A subset of 12 cane-resistant (scores 0 or 1) and 12 cane-susceptible (score 2 or 3) progeny from ‘Chardonnay’ × V. cinerea B9 were selected to maximize the number of progeny with recombination events around the Rda1 resistance locus. On August 29, 2013, three shoots on each field-grown vine was spray inoculated using a Preval handheld paint sprayer (Preval, IL, USA) immediately before sunset and enclosed in a moistened plastic bag to maintain surface wetness. The next morning (at 14 hpi), inoculated stem internode between the second and third unfurled leaf was collected, immediately stored in liquid nitrogen and transferred to the laboratory for RNA extraction.
|
study
| 100.0 |
Strand-specific, mRNA multiplexed libraries and RNA-Seq reads were processed as described above. EdgeR was used to determine normalized expression values as FPKM (Trapnell et al. 2010) and to determine DE transcripts between the resistant and susceptible samples (12 samples each) with a false-discovery rate (FDR) significance threshold of FDR ≤ 0.05, after Benjamini–Hochberg multiple comparison corrections. This experiment is referred to as the eQTL study in the following sections.
|
study
| 100.0 |
Analysis of variance (ANOVA) was used to determine the percentage of internode lesions on the California greenhouse experiment, using a factorial model for fixed effects plant genotype and experiment. For this, % internode lesions were converted to the mid-point of the percent range for each scale value (e.g., 45% for a score of “6”). ANOVA was performed using the MIXED procedure in SAS, with Kenward–Roger as the denominator degrees of freedom method (Littell et al. 1996). Homogeneity of variance across treatments was confirmed according to (Box et al. 1978). For significant effects (p < 0.05), differences among means were assessed based on the overlap of their 95% confidence intervals, and means without overlapping intervals were considered significantly different (Westfall et al. 1999).
|
study
| 100.0 |
Correlation between ratings of disease severity on dormant canes in NY field and mean % internode lesions in California greenhouse was determined by the CORR procedure in SAS, based on the Spearman rank-order correlation (non-parametric measure of association, based on the ranks of the data values).
|
study
| 99.94 |
In the field, lesions on dormant canes varied from absent (asymptomatic) to widespread with black, corky wood and canes that were visibly stunted (Fig. 1a). All parental genotypes showed few to no symptoms, having either a score of 0 (for one vine of ‘Chardonnay’, one vine of V. cinerea B9 and two vines of ‘Horizon’), or a score of 1 (three vines of ‘Chardonnay’, seven vines of V. cinerea B9, six vines of ‘Horizon’ and four vines of Illinois 547-1 displaying a small number of discrete cane lesions). While no parental plants showed scores of 2 or 3, these extremely susceptible phenotypes were observed for a proportion of progeny in all three F1 families (Fig. 2).Fig. 2Segregation of dormant cane symptoms and cluster symptoms in three F1 families. a ‘Chardonnay’ × V. cinerea B9, b ‘Horizon’ × V. cinerea B9 and c ‘Horizon’ × Illinois 547-1. Disease severity on canes was measured for 2 years using the following scale: (0) no Phomopsis symptoms observed; (1) light infection, small number of discrete lesions; (2) moderate infection, lesions coalescing, widespread; and (3) severe infection, lesions blackened, internode tissue corky and misshapen. On clusters, symptoms such as black superficial spots, shriveled berries and dry rachis were scored as present (1) or absent (0). Across all years, average cane severity was 0.75, 0.88, 0.75 and 1 for ‘Chardonnay’, V. cinerea B9, ‘Horizon’ and Illinois 547-1, respectively and 0 for the cluster-bearing parents ‘Chardonnay’ and ‘Horizon’
|
study
| 100.0 |
Segregation of dormant cane symptoms and cluster symptoms in three F1 families. a ‘Chardonnay’ × V. cinerea B9, b ‘Horizon’ × V. cinerea B9 and c ‘Horizon’ × Illinois 547-1. Disease severity on canes was measured for 2 years using the following scale: (0) no Phomopsis symptoms observed; (1) light infection, small number of discrete lesions; (2) moderate infection, lesions coalescing, widespread; and (3) severe infection, lesions blackened, internode tissue corky and misshapen. On clusters, symptoms such as black superficial spots, shriveled berries and dry rachis were scored as present (1) or absent (0). Across all years, average cane severity was 0.75, 0.88, 0.75 and 1 for ‘Chardonnay’, V. cinerea B9, ‘Horizon’ and Illinois 547-1, respectively and 0 for the cluster-bearing parents ‘Chardonnay’ and ‘Horizon’
|
study
| 100.0 |
Often, vines with symptoms on the canes also developed fruit symptoms. On immature clusters, black spots appeared on the berry surface and lesions on the rachis were also observed (Fig. 1c, d). After veraison, rachis lesions became dry and blackened, and berries became shriveled or split (Fig. 1c). Cluster symptoms were absent from female parents and were not possible to observe with the dioecious male parents V. cinerea B9 and Illinois 547-1. Among progeny, cane symptom scores correlated with the presence of cluster symptoms, with Pearson’s r of 0.92 and 0.76 in 2012–2013 and 2013–2014, respectively (Fig. 1d). The typical leaf spot symptom was rare among all families. Samples from symptomatic dormant canes incubated in humid conditions developed pycnidia that exude conidia (cirrhi), typical of D. ampelina. Conidia from these samples were successfully cultured on PDA plates, producing colonies with typical growth rings and cream colored cirrhi of pycnidia (Fig. 1b). Isolation of fungi was not successful from symptomatic berries.
|
study
| 100.0 |
A subset of six susceptible and four resistant progenies from the ‘Chardonnay’ × V. cinerea B9 cross were inoculated in the greenhouse to confirm that genotypes with susceptible phenotypes on dormant canes in NY field also expressed susceptible phenotypes (more typical stem and internode symptoms of Phomopsis cane and leaf spot) in an independent experiment (California). Our findings in the greenhouse were consistent with field observations. ANOVA detected significant differences in % internode lesions among genotypes (p < 0.0001). The two most susceptible genotypes in the field, 454077 and 454045, also had the highest levels of internode lesions in the greenhouse (Fig. 3). Similarly, the most resistant genotypes, which had field ratings of 0 or 1 (454053, 455035, 454058), were not significantly different from the parents, both of which had field ratings of 1. Non-inoculated plants developed no lesions, which suggests that symptoms were not due to remnant inoculum from the field, but from a different isolate of D. ampelina applied. The Spearman rank-order correlation between dormant cane field scores and the ranking of accessions based on percentage of symptomatic area on green stems was R2 = 0.78 with p value of 0.004. Consistent with field observations, leaf spots were rare in the greenhouse, even among genotypes with susceptible stems.Fig. 3Internode lesions (%) on the green stems of ten progenies from the F1 family of ‘Chardonnay’ × V. cinerea B9, after inoculation with D. ampelina in the greenhouse. The proportion of stem surface, spanning the four assessed internodes, covered by lesions was visually estimated at 30 d post-inoculation. ‘Parents’ are pooled values for Chardonnay and V. cinerea B9. Numbers at the base of each column are field ratings of disease severity on canes (on a scale of 0–3, Fig. 1). Each column is the mean of three observations, averaged across three replicate experiments (24 plants per genotype per experiment). Error bars are 95% confidence limits. Columns with overlapping error bars are not significantly different (p < 0.05; Tukey’s test)
|
study
| 100.0 |
Internode lesions (%) on the green stems of ten progenies from the F1 family of ‘Chardonnay’ × V. cinerea B9, after inoculation with D. ampelina in the greenhouse. The proportion of stem surface, spanning the four assessed internodes, covered by lesions was visually estimated at 30 d post-inoculation. ‘Parents’ are pooled values for Chardonnay and V. cinerea B9. Numbers at the base of each column are field ratings of disease severity on canes (on a scale of 0–3, Fig. 1). Each column is the mean of three observations, averaged across three replicate experiments (24 plants per genotype per experiment). Error bars are 95% confidence limits. Columns with overlapping error bars are not significantly different (p < 0.05; Tukey’s test)
|
study
| 100.0 |
To characterize the defense response of the resistant parent V. cinerea B9, we contrasted the expression of genes in V. cinerea B9 before (T0) and 48 h after (T2) inoculation with either D. ampelina or sterile water (mock). The mean number of sequencing reads obtained for this study was 10.3 million per replicate (supplementary Figure S1) or 30.9 million per treatment.
|
study
| 100.0 |
In inoculated V. cinerea B9, the 197 DE genes (T2 vs T0 at FDR ≤ 0.001) were unevenly distributed across 19 chromosomes (1.5–12.7% per chromosome), with inoculation enriching DE of genes on chromosomes 2, 9, 10, 16 and 18 (Fig. 4). Notably, genes on chromosome 15 showed a 2.9-fold repression of DE over time in treated samples, from 4.4% in mock inoculated to 1.5% in inoculated (Fig. 4). A greater number of genes (754) were DE in mock-inoculated vines, but had a less uneven distribution across 19 chromosomes (3.7–7.5% per chromosome).Fig. 4Chromosomal distribution of differentially expressed (DE) genes of V. cinerea B9 after inoculation with sterile water (mock DE, n = 751) or D. ampelina (inoculated DE, n = 290). For both treatments, genes with differential expression values between T0 (before inoculation) and T2 (48 h post-inoculation) were determined at FDR ≤ 0.001
|
study
| 100.0 |
Chromosomal distribution of differentially expressed (DE) genes of V. cinerea B9 after inoculation with sterile water (mock DE, n = 751) or D. ampelina (inoculated DE, n = 290). For both treatments, genes with differential expression values between T0 (before inoculation) and T2 (48 h post-inoculation) were determined at FDR ≤ 0.001
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There were 122 shared DE genes between mock and D. ampelina inoculated samples. Inoculated-exclusive DE genes were strongly enriched in pathways associated with ethylene signaling and phenylpropanoid biosynthesis and significantly enriched in pathways associated with anthocyanin biosynthesis and nitrogen metabolism (Supplementary Table S2). Out of the 75 inoculated-exclusive DE genes, 56 genes (74.6%) were down-regulated 48 h after inoculation (Table 2), including several ethylene-responsive transcription factors and auxin-related proteins. Among others, pathogenesis proteins, peroxidases, stilbene synthase, tropinone reductase, dirigent protein and the cytochrome P450 hydroxylase CYP86A1 were up-regulated at 48 h after inoculation.Table 2Vitis cinerea B9 transcripts with Diaporthe ampelina inoculation-exclusive differential expression (DE) genes at 2 days post-inoculation, using false-discovery rate (FDR) ≤ 0.001GeneIDFunctional annotationFDRlogFCVIT_03s0063g00460Ethylene-responsive transcription factor ERF1095.0 × 10−10− 8.01VIT_07s0005g05910Auxin-binding protein ABP195.2 × 10−4− 6.30VIT_11s0016g00660DREB sub A-5 of ERF/AP2 transcription factor2.7 × 10−10− 5.34VIT_16s0013g01060Ethylene-responsive transcription factor ERF1051.2 × 10−4− 4.29VIT_16s0013g01030Ethylene-responsive transcription factor ERF1053.4 × 10−6− 3.63VIT_09s0002g02030Pyruvoyl-dependent arginine decarboxylase7.9 × 10−4− 3.60VIT_09s0002g080602-Hydroxyacid dehydrogenases, D-isomer specific8.2 × 10−7− 3.56VIT_16s0013g00950Ethylene-responsive transcription factor ERF1051.9 × 10−7− 3.53VIT_06s0009g03670F-box family protein1.0 × 10−5− 3.45VIT_08s0007g08520Unknown protein9.6 × 10−14− 3.31VIT_03s0180g00210Myb domain protein R12.5 × 10−5− 3.29VIT_14s0066g02350Galactinol synthase9.6 × 10−8− 3.26VIT_01s0127g00700Unknown protein1.1 × 10−15− 3.24VIT_07s0005g01140Unknown protein6.3 × 10−4− 3.21VIT_13s0064g01110No hit5.3 × 10−7− 3.17VIT_16s0013g01050Ethylene-responsive transcription factor ERF1051.2 × 10−7− 3.15VIT_06s0009g01620Harpin-induced protein3.4 × 10−6− 3.11VIT_16s0013g00990Ethylene-responsive transcription factor ERF1053.3 × 10−7− 3.10VIT_06s0080g01090CCR4-NOT transcription complex subunit 7/88.4 × 10−11− 3.10VIT_11s0016g01810Unknown protein1.8 × 10−13− 3.10VIT_18s0001g073202-Oxoglutarate/malate carrier protein, Mitochondrial2.4 × 10−9− 3.08VIT_12s0134g00240Avr9/Cf-9 rapidly elicited protein 201.3 × 10−8− 2.98VIT_14s0081g00520ERF128.8 × 10−5− 2.93VIT_06s0004g04180Zinc finger (C2H2 type) protein (ZAT11)6.2 × 10−4− 2.82VIT_18s0001g09230Salt-tolerance zinc finger2.3 × 10−5− 2.79VIT_16s0013g00970Ethylene-responsive element-binding factor 53.5 × 10−7− 2.72VIT_02s0025g02490Unknown protein2.0 × 10−4− 2.70VIT_16s0013g00980Ethylene-responsive transcription factor ERF1055.3 × 10−4− 2.67VIT_19s0093g005509-Cis-epoxycarotenoid dioxygenase 21.4 × 10−4− 2.59VIT_09s0054g01410Beta-amyrin synthase7.7 × 10−9− 2.59VIT_17s0000g01630Calmodulin CML374.7 × 10−4− 2.59VIT_12s0028g03270Ethylene-responsive transcription factor 91.5 × 10−5− 2.54VIT_18s0001g11170Myb domain protein 739.2 × 10−6− 2.54VIT_12s0134g00170No hit2.5 × 10−5− 2.53VIT_16s0013g01000Ethylene-responsive transcription factor ERF1058.2 × 10−4− 2.51VIT_02s0012g02820Geraniol 10-hydroxylase2.1 × 10−4− 2.46VIT_07s0255g00020OBF-binding protein 12.8 × 10−4− 2.44VIT_08s0105g00190U-box domain-containing protein1.5 × 10−5− 2.43VIT_19s0014g02240Ethylene-responsive element-binding factor 46.7 × 10−9− 2.43VIT_18s0122g00300Unknown protein1.2 × 10−5− 2.42VIT_05s0077g01970Zinc finger (C3HC4-type ring finger)3.6 × 10−9− 2.39VIT_09s0002g08030Arogenate dehydrogenase isoform 24.5 × 10−5− 2.34VIT_01s0011g04550Unknown protein1.1 × 10−6− 2.32VIT_18s0001g06560No hit8.1 × 10−9− 2.30VIT_05s0020g04570CBL-interacting protein kinase 7 (CIPK7)2.7 × 10−5− 2.20VIT_03s0038g02140Auxin transporter protein 25.4 × 10−4− 2.19VIT_18s0122g00980Glucan endo-1,3-beta-glucosidase 7 precursor2.5 × 10−4− 2.16VIT_17s0000g09270MATE efflux family protein1.5 × 10−5− 2.16VIT_00s0267g00030Unknown2.9 × 10−4− 2.15VIT_18s0166g00190U-box domain-containing protein4.1 × 10−4− 2.15VIT_00s0218g00140Anthocyanidine rhamnosyl-transferase4.8 × 10−4− 2.14VIT_18s0001g09910l-Asparaginase3.7 × 10−7− 2.14VIT_02s0012g02810CYP76C41.2 × 10−4− 2.13VIT_15s0048g02070BON2-associated protein (BAP2)3.4 × 10−4− 2.09VIT_16s0050g01580UDP-glucose: anthocyanidin 5,3-O-glucosyltransferase8.2 × 10−4− 2.06VIT_03s0063g00830Carboxyesterase 5 CXE51.7 × 10−4− 2.04VIT_15s0107g00550Tetratricopeptide repeat domain male sterility MS51.2 × 10−52.14VIT_16s0100g00930Stilbene synthase 21.5 × 10−42.61VIT_00s0229g00190Inositol 2-dehydrogenase like protein1.4 × 10−62.66VIT_16s0039g01300Phenylalanine ammonia-lyase (Vitis vinifera)4.3 × 10−42.73VIT_16s0100g00810Stilbene synthase (Vitis vinifera)3.3 × 10−72.82VIT_05s0077g01560Pathogenesis protein 10.3 (Vitis quinquangularis)3.8 × 10−93.12VIT_16s0100g00900Stilbene synthase (Vitis pseudoreticulata)3.5 × 10−73.22VIT_16s0100g00860Chalcone synthase1.4 × 10−73.48VIT_16s0100g01030Stilbene synthase (Vitis quinquangularis)1.2 × 10−53.54VIT_05s0077g01550Pathogenesis protein 10.3 (Vitis quinquangularis)8.9 × 10−53.56VIT_18s0001g06850Peroxidase GvPx2b class III1.1 × 10−73.67VIT_05s0077g01530Pathogenesis protein 10 (Vitis vinifera)1.7 × 10−83.71VIT_05s0077g01570Pathogenesis protein 10 (Vitis vinifera)3.2 × 10−123.80VIT_16s0100g01150Stilbene synthase (Vitis vinifera)1.4 × 10−64.13VIT_08s0007g00920Tropinone reductase8.8 × 10−54.13VIT_04s0069g00730Glutamate receptor protein7.3 × 10−44.18VIT_07s0031g01680CYP86A15.1 × 10−44.34VIT_06s0004g01020Dirigent protein4.3 × 10−54.62VIT_16s0100g00940Stilbene synthase 3 (Vitis sp. cv. ‘Norton’)2.9 × 10−44.94Fifty-six ###genes with negative log2 fold change (logFC) were down-regulated after inoculation (logFC < − 2), and nineteen genes with positive logFC were up-regulated after inoculation (logFC > 2)
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The high-density genetic maps used for this analysis were derived from genotyping by sequencing using the pseudo-testcross approach. Since the 12X.0 version of the PN40024 reference genome was used, markers have both physical and genetic positions (Hyma et al. 2015).
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| 99.94 |
Two major loci located on chromosomes 15 and 7 from the V. cinerea B9 and ‘Horizon’ parents, respectively, showed dominant effects and significantly predicted both the severity and incidence of cane and cluster symptoms, respectively, for all years and families tested (Table 3). Here, we refer to these V. cinerea B9 and ‘Horizon’ loci as Rda1 and Rda2, respectively. For all crosses used in this study, vines with either the Rda1 or Rda2 resistance allele had either no symptoms or small, discrete lesions (scores 0 or 1), while vines with both susceptible alleles showed moderate to severe symptoms (scores 2–3). On the Illinois 547-1 map, two other minor QTL were significant only in the 2011 evaluation of dormant canes and explained much less of the phenotypic variance (3.2 and 3.5%) than Rda1 or Rda2 (28.4 and 24.8%) (Table 3). No QTL was identified from ‘Chardonnay’.Table 3QTL mapping statisticsFamilyParentChraPhenotypeYearPeak Markera (cM)Left Markera (cM)Right Markera (cM)LODPVEc (%)‘Chardonnay’ × V. cinerea B9V. cinerea B915Cane2012S15_19560016 (62.2)S15_19299979 (61.4)S15_19591520 (63.7)51.479.82013S15_19560016 (62.2)S15_19299979 (61.4)S15_19591520 (63.7)44.275.2Cluster2013S15_19591520 (63.7)S15_18780806 (57.1)S15_20031941 (66.8)*16.573.02014S15_19560016 (62.2)S15_18780806 (57.1)S15_20031941 (66.8)*10.560.7‘Horizon’ × V. cinerea B9‘Horizon’7Cane2012S7_2768585 (15.2)S7_1087848 (10.0)S7_3855744 (19.1)50.441.12013S7_3127568 (15.5)S7_1087848 (10.0)S7_3855744 (19.1)29.148.0Cluster2013S7_3127568 (15.5)S7_1087848 (10.0)S7_4952429 (24.7)30.622.82014S7_1860119 (13.9)S7_1087848 (10.0)S7_4952429 (24.7)25.111.2V. cinerea B915Cane2012S15_19591538 (51.4)S15_19560016 (50.6)S15_19637245 (53.1)*56.151.02013S15_19591538 (51.4)S15_19560016 (50.6)S15_19637245 (53.1)*32.256.3Cluster2013S15_19591538 (51.4)S15_19560016 (50.6)S15_19637245 (53.1)*30.120.02014S15_19637245 (53.1)S15_19560016 (50.6)S15_19637245 (53.1)*26.520.4‘Horizon’ × Illinois 547-1‘Horizon’7Cane2011S7_2000903 (6.5)S7_1459378 (4.5)S7_2409624 (7.7)30.324.82012S7_1912889 (5.6)S7_1459378 (4.5)S7_2409624 (7.7)58.445.5Illinois 547-11Cane2011S1_3046182 (11.3)S1_1170106 (3.8)S1_4279265 (14.5)3.63.222011S2_5852870 (34.5)S2_2340804 (12.0)S2_7231845 (40.5)4.73.4152011S15_19300044 (49.2)S15_19053446 (46.1)S15_19591538 (54.7)34.228.42012S15_19300044 (49.2)S15_19300044 (49.2)S15_19591538 (54.7)58.646.1Loci associated with Phomopsis cane and berry symptoms were identified by multiple QTL mapping on parental maps for three familiesaChromosome (Chr) and marker positions correspond to the physical location in the 12X.0 PN40024 Vitis vinifera reference genome. Markers are reported in the format S(chromosome)_(position in bp). Left and right markers correspond to the closest marker to the borders of a 1.5 LOD interval. An asterisk (*) indicates the last marker of the mapbLOD threshold was determined by permutation test (1000), at α = 0.05, and ranged from 2.90 to 3.14cPVE refers to the percentage of variance explained by the locus
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aChromosome (Chr) and marker positions correspond to the physical location in the 12X.0 PN40024 Vitis vinifera reference genome. Markers are reported in the format S(chromosome)_(position in bp). Left and right markers correspond to the closest marker to the borders of a 1.5 LOD interval. An asterisk (*) indicates the last marker of the map
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| 99.9 |
According to the physical position of flanking markers, the smallest supported interval for the Rda1 locus is located between 19.3 and 19.6 Mbp of chromosome 15 (with higher LOD of 51.4 and 58.6, Table 3), and the smallest supported interval for the Rda2 locus is located between 1.5 and 2.4 Mbp of chromosome 7 for the result with higher LOD (Table 3, LOD 58.4). There are 39 annotated genes within the 300 kb supported interval for the Rda1 locus, which codes for five NB-LRR proteins (Grimplet et al. 2012) that are potentially associated with plant–pathogen interactions.
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Based on a subset of 94 individuals, three SSR markers, VVIB22 (Merdinoglu et al. 2005), VrZAG62 (This et al. 2004), and VVMD7 (Bowers et al. 1996) located on chromosome 7, near the Rda2 locus, were confirmed to be linked to Rda2 (Table 4). SC8_0040_088 (Jaillon et al. 2007) was the only SSR marker near Rda1 in the PN40024 reference (18.9 Mbp), but was homozygous in the resistant parent V. cinerea B9 (358 bp) and thus non-informative in the progeny.Table 4SSR allele sizes in linkage with the Rda2 locusSSR markerPhysical location (Mbp)‘Horizon’V. cinerea B9Allele size (bp)p valueAllele size (bp)p valueVVIB223.10157/1391.8 × 10−12144/1600.068VrZAG621.78180/2021.8 × 10−12174/1880.650VVMD71.17237/2351.2 × 10−11231/231naAlleles associated with resistance to Phomopsis cane lesions from ‘Horizon’ are indicated in bold. Linkage was determined by χ2 test over a subset of 66 progeny from ‘Horizon’ × Vitis cinerea B9
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We used an eQTL approach to further investigate the association between expression of candidate genes and the resistance locus. For this, 12 resistant and 12 susceptible vines from the ‘Chardonnay’ × V. cinerea B9 progeny were sampled, 14 of which exhibited recombination within 15 cM of the Rda1 locus (Fig. 5). The total number of quality reads was 32.01 million per treatment (± Rda1 allele), with a sample mean and median of 2.67 million reads and 2.20 million reads per progeny, respectively (Supplementary Figure S1).Fig. 5Genotypes on chromosome 15 of the Vitis cinerea B9 map for individuals selected for RNA-Seq. Resistant (upper) and susceptible (lower) progeny showed genotype segregation at the Rda1 locus. The marker with highest LOD score is indicated. White and black dots indicate the allelic states AAxBA and AAxAB, respectively
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| 100.0 |
Genotypes on chromosome 15 of the Vitis cinerea B9 map for individuals selected for RNA-Seq. Resistant (upper) and susceptible (lower) progeny showed genotype segregation at the Rda1 locus. The marker with highest LOD score is indicated. White and black dots indicate the allelic states AAxBA and AAxAB, respectively
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| 99.94 |
We found 16 DE genes between resistant and susceptible progeny 14 h after inoculation, at FDR ≤ 0.05, including three NB-LRR genes on chromosome 15 (Supplementary Table S3). Expression was significantly predicted by alleles at the Rda1 locus for 6 of these 16 genes: a mannitol dehydrogenase gene and the three aforementioned NB-LRRs on chromosome 15 were up-regulated in susceptible vines, while a cytochrome P450 monooxygenase gene (CYP78A3p) and an auxin-responsive protein IAA17 gene were up-regulated in resistant vines (Table 5). The NB-LRRs VIT_15s0046g02730 and VIT_15s0046g02800 located at 19.45 and 19.53 Mbp, respectively, were the only two eQTL located within the Rda1 interval in the PN40024 12X.0 reference genome.Table 5Differential expression (DE) and expression QTL (eQTL) mapping statisticsGeneGene chraGene position (bp)aGene functional annotationlogFCbDE p valueeQTL LODeQTL LOD ThrceQTL PVEdeQTL effectVIT_15s0046g027301519,454,696–19,457,671PRF disease resistance protein2–40.0016.33.570.16.06VIT_15s0021g00120159,388,654–9,389,448RPP13 recognition of Peronospora parasitica 1325.36 × 10−115.13.662.719.9VIT_15s0046g028001519,528,135–19,530,195PRF disease resistance protein6.50.0054.83.460.31.92VIT_00s0346g00110Un24,788,096–24,791,922Mannitol dehydrogenase4.50.0273.13.044.86.30VIT_15s0048g029001517,005,384–17,007,131Cytochrome P450 monooxygenase CYP78A3p− 3.40.0293.12.645.0− 5.90VIT_09s0002g0516094,853,689–4,862,025Auxin-responsive protein IAA17− 2.40.0293.63.050.1− 12.6Six genes showed association between transcription levels and the Rda1 locus, located between 19.3 and 19.6 Mbp of chromosome 15Genetic maps were used for multiple QTL mapping of transcription levels (FPKM) of differentially expressed genes on a subset of 24 progeny from ‘Chardonnay’ × V. cinerea B9aChr and gene positions correspond to the physical location in the 12X.0 PN40024 Vitis vinifera reference genome; chr Un corresponds to the unassembled pseudo chromosomeblogFC corresponds to the log2 fold changecLOD Thr (threshold) was determined by permutation test (10,000) at α = 0.05dPVE refers to the percentage of transcript variance explained by the Rda1 locus
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| 100.0 |
This study started with observations of Phomopsis symptoms among segregating families in different environments and proceeded to genetic mapping of loci controlling the resistance phenotype. Within the Rda1 locus, we used transcriptome screening to narrow down candidates to two NB-LRR loci, providing the first clue for the molecular resistance mechanism for this pathogen.
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| 100.0 |
First, we found complementary evidence from experiments in the field and greenhouse that there is genetic resistance to Phomopsis cane and leaf spot among plant genotypes representative of segregating families, based on observations of symptoms on different grapevine organs (canes, shoots, and clusters). Our findings of Phomopsis susceptibility in powdery mildew-resistant breeding materials makes it clear that cessation of fungicide use to minimize the latter could exacerbate the former. Understanding the genetics of resistance to Phomopsis cane and leaf spot on these organs could facilitate better strategies for its management in diverse environments, such as the east and west coasts of the USA, where the pathogens of both diseases are a problem.
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| 99.94 |
Segregation ratios observed in the three hybrid families suggested the presence of one major dominant locus in ‘Chardonnay’ × V. cinerea B9 (1 resistant:1 susceptible) and at least two major dominant loci in ‘Horizon’ × V. cinerea B9 (3 resistant:1 susceptible) and ‘Horizon’ × Illinois 547-1 (V. rupestris B38 × V. cinerea B9). This observation was corroborated by QTL mapping, where the loci Rda1 and Rda2 were found in V. cinerea B9 and ‘Horizon’, respectively. Co-localization of loci obtained from cane and cluster phenotypes suggests that resistance in both tissues was due to the same loci. Therefore, we used the Rda1 and Rda2 designations for both phenotypes.
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| 100.0 |
Both Rda1 and Rda2 loci showed major dominant effects that suggested qualitative resistance, providing protection against disease symptoms. As a consequence, the molecular markers provided for Rda2 can be used directly for marker-assisted selection of resistant vines at the seedling stage or for stacking Phomopsis resistance alleles along with resistance to major grapevine pathogens. For Rda1, readily assayed markers for MAS can be obtained from the GBS-SNPs provided in Table 3, as described previously (Yang et al. 2016).
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| 100.0 |
Even though ‘Chardonnay’ disease severity was similar to that of V. cinerea B9 in both the greenhouse and field, and to ‘Horizon’ in the field, we were not able to identify resistance loci from ‘Chardonnay’. If ‘Chardonnay’ resistance is quantitative, our experiment may not have had enough statistical power to detect minor effect QTL. Among ‘Chardonnay’ × V. cinerea B9 F1 progeny, segregation of ‘Chardonnay’ loci can only be observed among progeny with Rda1 susceptible alleles, reducing the effective size of the population to fewer than 100 individuals. While ‘Chardonnay’ resistance could instead be recessively inherited, the rare presence of extremely susceptible phenotypes in related V. vinifera cultivars argues against that possibility.
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The extremely susceptible phenotype observed in progeny that did not inherit a resistance allele was not typical of Phomopsis cane and leaf spot symptoms or fruit symptoms seen in commercial vineyards. This may be expected, since cultivated and bred grapes have been subjected to selection, which may have purged these extremely susceptible phenotypes. As an example, in cultivated grapevines, D. ampelina fruit infections initiated at the pedicel are typically latent during most of summer and symptoms do not appear until harvest, when berries rot and black pycnidia form (Wilcox et al. 2015). At our field site, symptoms were unusually severe, with black lesions appearing on the berries and rachises drying even before veraison. In some cases, berries did not even expand, but instead remained stunted and became necrotic. We did not recover D. ampelina from symptomatic clusters, as we did from the canes; nonetheless, the correlation between cluster and cane symptoms was evident. The cane symptoms observed in the field started out as lesions on the green stems; levels of cane symptoms in the field and internode lesions in the greenhouse on genotypes were positively correlated under both conditions.
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At 48 hpi with D. ampelina, the resistant parent V. cinerea B9 was symptomless, with a transcriptome showing a complex profile with elements typical of immune responses mediated by NB-LRR and repression of antagonistic pathways. Up-regulated genes, such as pathogenesis-related proteins along with salicylic acid signaling genes, are consistent with defense responses to biotrophic pathogens. Other mechanisms of defense were also present, such as strengthening of physical defenses by the up-regulation of peroxidase class III, cytochrome P450 hydrolase CYP86A1 gene, involved in the biosynthesis of suberin (Hofer et al. 2008), or by up-regulation of a dirigent protein gene, involved in tissue lignification (Davin and Lewis 2000). Other up-regulated genes were associated with the production of defense-related secondary metabolites, such as stilbene synthases or tropinone reductase, related to alkaloid metabolism (Drager 2006). Down-regulation of genes involved in the ethylene signaling pathway as well as the auxin signaling pathway is required to activate the antagonistic salicylic acid (SA) pathway (Chang et al. 2015; Kazan and Manners 2009) and is also consistent with a biotrophic immune response. A possible future experiment could include validation of these expression differences by qRT-PCR.
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| 100.0 |
High-density genetic maps, derived from sequencing small fragments and alignment to the V. vinifera PN40024 reference genome, allowed the immediate localization of flanking SNP markers in the physical map without the need for BAC libraries or further sequencing. For Rda1, the smallest supported interval of 300 kb between 19.3 and 19.6 Mbp on chromosome 15, determined in the result with higher LOD (Table 3, LOD 51.4 and 58.6), contains a cluster of NB-LRR genes (Grimplet et al. 2012). As described in other pathosystems, NB-LRR genes are candidates for qualitative resistance to a hemibiotrophic pathogen, such as D. ampelina. For Rda2, the smallest supported interval had less resolution, being located between 1.46 and 2.41 Mbp of chromosome 7 for the result with higher LOD (Table 3, LOD 58.4). This larger region of approximately 950 kb has 134 annotated genes (Grimplet et al. 2012) and no obvious candidate.
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| 100.0 |
To further investigate the Rda1 locus, 24 full sibling progenies were used to delineate candidate resistance genes with an eQTL approach at 14 hpi. To increase the statistical power of this analysis, we followed some simple steps: first, we used the saturated genetic maps and the Rda1 locus position to identify vines with nearby recombination. These vines were sampled and used for construction of RNA-Seq libraries to provide better mapping resolution by maximizing the recombination events within and near the locus. Then, we focused the eQTL analysis by selecting those transcripts that were DE between vines with resistant and susceptible phenotypes, reducing the number of eQTL tests from 30,034 annotated PN40024 transcripts to only 16 DE transcripts (Supplementary Table S3). An eQTL scan for loci predicting expression of these 16 genes identified two candidate NB-LRR genes (VIT_15s0046g02730 and VIT_15s0046g02800) as differentially expressed and significantly associated to the Rda1 locus. We also identified one NB-LRR gene physically distant on chromosome 15 (according to the PN40024 reference) and three genes on other chromosomes regulated by the Rda1 locus early after D. ampelina inoculation: a mannitol dehydrogenase, the cytochrome P450 monooxygenase CYP78A3p and the auxin-responsive protein IAA17 (Table 5). While the eQTL hits located on Rda1 can point out two candidate R-genes for this locus, hits from other loci could be related to reactions triggered by ETI.
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| 100.0 |
The elevated number of NB-LRR gene transcripts up-regulated in the susceptible progeny compared with the resistant progeny suggests that susceptibility and not resistance is mediated by the action of NB-LRR genes, which may facilitate the necrotrophic phase of the hemibiotrophic fungus. This is reminiscent of the wheat NB-LRR protein Tsn1, which confers sensitivity to ToxA from the necrotrophic fungi Stagonospora nodorum and Pyrenophora tritici-repentis (Faris et al. 2010). Alternatively, sequence divergence between the resistance allele and the reference transcriptome derived from V. vinifera ‘Pinot noir’ may have resulted in a misalignment of reads, resulting in lower FPKM values for the resistance allele. To elucidate this issue, a de novo transcriptome for the resistant parent was constructed using the DE study total reads, eQTL analysis was repeated and associated transcripts were annotated. We obtained the same eQTL as reported when using the reference genome; specifically, three of the four most significant DE genes were NB-LRR genes 80- to 410-fold up-regulated in susceptible versus resistant progeny (data not shown).
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| 100.0 |
Another explanation is that the time point used was not representative of the plant response. For this, expression differences should be validated using qRT-PCR at several time points between inoculation and the appearance of symptoms on the susceptible vines. Moreover, a comparison of the molecular response between resistant and susceptible progenies to the biotrophic and necrotrophic phases of the pathogen would help to elucidate the hypothesis of susceptibility mediated by NB-LRR genes as stated above.
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study
| 100.0 |
In RNA-Seq experiments, we used 14 hpi for field (eQTL experiment) and 48 hpi for growth chamber inoculations (DE experiment) to study the early response of the plant. As described, the beginning of D. ampelina life cycle suggests a biotrophic phase (Willison et al. 1965) that, in a successful interaction, develops lesions after 7 days, which are typically associated with its necrotrophic phase (Wilcox et al. 2015). This suggests that both experiments were conducted during the biotrophic phase of the pathogen, which is consistent with the absence of symptoms at the time of sample collection, and the plant transcriptome response activated toward a biotrophic response. In other pathosystems, such as the hemibiotrophic pathogen Phytophthora infestans in potato, 48 hpi also showed consistency between the absence of symptoms and a transcriptome response associated with a plant–biotroph pathogen interaction (Zuluaga et al. 2016).
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| 100.0 |
The set of DE genes identified in the eQTL study of progeny vines differs from that in the DE study of the V. cinerea B9 parent. This is expected as consequences of distinct experimental designs. Differences in the V. cinerea B9 DE study reflected changes in expression between time points (0 and 48 hpi) for a single parental genotype. In contrast, differential expression in the eQTL study was among different genotypes (full siblings) at a single time point (14 hpi), obscuring whether genes were constitutively or dynamically differential. As the resistant Rda1 allele is dominant, observing the effect of the susceptible allele was only possible in the eQTL study, in which the two Rda1 alleles segregate, but not in the V. cinerea B9 DE study. Moreover, in the eQTL experiment, ‘Chardonnay’ alleles are also present, which can change the transcriptome profiles related to the V. cinerea B9 DE study. Regardless of these differences, both approaches suggest that NB-LRR gene-mediated host responses may be critical in determining the outcome of infection.
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| 100.0 |
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