The grapevine LysM receptor-like kinase VvLYK5-1 recognizes chitin oligomers through its association with VvLYK1-1.

The establishment of defense reactions to protect plants against pathogens requires the recognition of invasion patterns (IPs), mainly detected by plasma membrane-bound pattern recognition receptors (PRRs). Some IPs, also termed elicitors, are used in several biocontrol products that are gradually being developed to reduce the use of chemicals in agriculture. Chitin, the major component of fungal cell walls, as well as its deacetylated derivative, chitosan, are two elicitors known to activate plant defense responses. However, recognition of chitooligosaccharides (COS) in Vitis vinifera is still poorly understood, hampering the improvement and generalization of protection tools for this important crop. In contrast, COS perception in the model plant Arabidopsis thaliana is well described and mainly relies on a tripartite complex formed by the cell surface lysin motif receptor-like kinases (LysM-RLKs) AtLYK1/CERK1, AtLYK4 and AtLYK5, the latter having the strongest affinity for COS. In grapevine, COS perception has for the moment only been demonstrated to rely on two PRRs VvLYK1-1 and VvLYK1-2. Here, we investigated additional players by overexpressing in Arabidopsis the two putative AtLYK5 orthologs from grapevine, VvLYK5-1 and VvLYK5-2. Expression of VvLYK5-1 in the atlyk4/5 double mutant background restored COS sensitivity, such as chitin-induced MAPK activation, defense gene expression, callose deposition and conferred non-host resistance to grapevine downy mildew (Erysiphe necator). Protein-protein interaction studies conducted in planta revealed a chitin oligomer-triggered interaction between VvLYK5-1 and VvLYK1-1. Interestingly, our results also indicate that VvLYK5-1 mediates the perception of chitin but not chitosan oligomers showing a part of its specificity.

Grapevine Rpv3-, Rpv10- and Rpv12-mediated defense responses against Plasmopara viticola and the impact of their deployment on fungicide use in viticulture.

BACKGROUND: The high susceptibility of European grapevine cultivars (Vitis vinifera) to downy mildew (Plasmopara viticola) leads to the intensive use of fungicides in viticulture. To reduce this input, breeding programs have introgressed resistance loci from wild Vitis species into V. vinifera, resulting in new fungus-resistant grapevine cultivars (FRC). However, little is known about how these different resistance loci confer resistance and what the potential reduction in fungicide applications are likely to be if these FRCs are deployed. To ensure a durable and sustainable resistance management and breeding, detailed knowledge about the different defense mechanisms mediated by the respective Rpv (Resistance to P. viticola) resistance loci is essential. RESULTS: A comparison of the resistance mechanisms mediated by the Rpv3-1, Rpv10 and/or Rpv12-loci revealed an early onset of programmed cell death (PCD) at 8 hours post infection (hpi) in Rpv12-cultivars and 12 hpi in Rpv10-cultivars, whereas cell death was delayed in Rpv3-cultivars and was not observed until 28 hpi. These temporal differences correlated with an increase in the trans-resveratrol level and the formation of hydrogen peroxide shortly before onset of PCD. The differences in timing of onset of Rpv-loci specific defense reactions following downy mildew infection could be responsible for the observed differences in hyphal growth, sporulation and cultivar-specific susceptibility to this pathogen in the vineyard. Hereby, Rpv3- and Rpv12/Rpv3-cultivars showed a potential for a significant reduction of fungicide applications, depending on the annual P. viticola infection pressure and the Rpv-loci. Furthermore, we report on the discovery of a new P. viticola isolate that is able to overcome both Rpv3- and Rpv12-mediated resistance. CONCLUSION: This study reveals that differences in the timing of the defense reaction mediated by the Rpv3-, Rpv10- and Rpv12-loci, result in different degrees of natural resistance to downy mildew in field. Vineyard trials demonstrate that Rpv12/Rpv3- and Rpv3-cultivars are a powerful tool to reduce the dependence of grape production on fungicide applications. Furthermore, this study indicates the importance of sustainable breeding and plant protection strategies based on resistant grapevine cultivars to reduce the risk of new P. viticola isolates that are able to overcome the respective resistance mechanism.

Transcriptional profiling reveals multiple defense responses in downy mildew-resistant transgenic grapevine expressing a TIR-NBS-LRR gene located at the MrRUN1/MrRPV1 locus.

Grapevine downy mildew (DM) is a destructive oomycete disease of viticulture worldwide. MrRPV1 is a typical TIR-NBS-LRR type DM disease resistance gene cloned from the wild North American grapevine species Muscadinia rotundifolia. However, the molecular basis of resistance mediated by MrRPV1 remains poorly understood. Downy mildew-susceptible Vitis vinifera cv. Shiraz was transformed with a genomic fragment containing MrRPV1 to produce DM-resistant transgenic Shiraz lines. Comparative transcriptome analysis was used to compare the transcriptome profiles of the resistant and susceptible genotypes after DM infection. Transcriptome modulation during the response to P. viticola infection was more rapid, and more genes were induced in MrRPV1-transgenic Shiraz than in wild-type plants. In DM-infected MrRPV1-transgenic plants, activation of genes associated with Ca2+ release and ROS production was the earliest transcriptional response. Functional analysis of differentially expressed genes revealed that key genes related to multiple phytohormone signaling pathways and secondary metabolism were highly induced during infection. Coexpression network and motif enrichment analysis showed that WRKY and MYB transcription factors strongly coexpress with stilbene synthase (VvSTS) genes during defense against P. viticola in MrRPV1-transgenic plants. Taken together, these findings indicate that multiple pathways play important roles in MrRPV1-mediated resistance to downy mildew.

Arabidopsis downy mildew effector HaRxLL470 suppresses plant immunity by attenuating the DNA-binding activity of bZIP transcription factor HY5.

The oomycete pathogen Hyaloperonospora arabidopsidis delivers diverse effector proteins into host plant cells to suppress the plant's innate immunity. In this study, we investigate the mechanism of action of a conserved RxLR effector, HaRxLL470, in suppressing plant immunity. Genomic, molecular and biochemical analyses were performed to investigate the function of HaRxLL470 and the mechanism of the interaction between HaRxLL470 and the target host protein during H. arabidopsidis infection. We report that HaRxLL470 enhances plant susceptibility to H. arabidopsidis isolate Noco2 by interacting with the host photomorphogenesis regulator protein HY5. Our results demonstrate that HY5 is not only an important component in the regulation of light signalling, but also positively regulates host plant immunity against H. arabidopsidis by transcriptional activation of defense-related genes. We show that the interaction between HaRxLL470 and HY5 compromises the function of HY5 as a transcription factor by attenuating its DNA-binding activity. The present study demonstrates that HY5 positively regulates host plant defense against H. arabidopsidis whereas HaRxLL470, a conserved RxLR effector across oomycete pathogens, enhances pathogenicity by interacting with HY5 and suppressing transcriptional activation of defense-related genes.

NAD+ cleavage activity by animal and plant TIR domains in cell death pathways.

SARM1 (sterile alpha and TIR motif containing 1) is responsible for depletion of nicotinamide adenine dinucleotide in its oxidized form (NAD+) during Wallerian degeneration associated with neuropathies. Plant nucleotide-binding leucine-rich repeat (NLR) immune receptors recognize pathogen effector proteins and trigger localized cell death to restrict pathogen infection. Both processes depend on closely related Toll/interleukin-1 receptor (TIR) domains in these proteins, which, as we show, feature self-association-dependent NAD+ cleavage activity associated with cell death signaling. We further show that SARM1 SAM (sterile alpha motif) domains form an octamer essential for axon degeneration that contributes to TIR domain enzymatic activity. The crystal structures of ribose and NADP+ (the oxidized form of nicotinamide adenine dinucleotide phosphate) complexes of SARM1 and plant NLR RUN1 TIR domains, respectively, reveal a conserved substrate binding site. NAD+ cleavage by TIR domains is therefore a conserved feature of animal and plant cell death signaling pathways.

Rpv3-1 mediated resistance to grapevine downy mildew is associated with specific host transcriptional responses and the accumulation of stilbenes.

BACKGROUND: European grapevine cultivars (Vitis vinifera spp.) are highly susceptible to the downy mildew pathogen Plasmopara viticola. Breeding of resistant V. vinifera cultivars is a promising strategy to reduce the impact of disease management. Most cultivars that have been bred for resistance to downy mildew, rely on resistance mediated by the Rpv3 (Resistance to P. viticola) locus. However, despite the extensive use of this locus, little is known about the mechanism of Rpv3-mediated resistance. RESULTS: In this study, Rpv3-mediated defense responses were investigated in Rpv3+ and Rpv3- grapevine cultivars following inoculation with two distinct P. viticola isolates avrRpv3+ and avrRpv3-, with the latter being able to overcome Rpv3 resistance. Based on comparative microscopic, metabolomic and transcriptomic analyses, our results show that the Rpv3-1-mediated resistance is associated with a defense mechanism that triggers synthesis of fungi-toxic stilbenes and programmed cell death (PCD), resulting in reduced but not suppressed pathogen growth and development. Functional annotation of the encoded protein sequence of genes significantly upregulated during the Rpv3-1-mediated defense response revealed putative roles in pathogen recognition, signal transduction and defense responses. CONCLUSION: This study used histochemical, transcriptomic and metabolomic analyses of Rpv3+ and susceptible cultivars inoculated with avirulent and virulent P. viticola isolates to investigate mechanism underlying the Rpv3-1-mediated resistance response. We demonstrated a strong correlation between the expressions of stilbene biosynthesis related genes, the accumulation of fungi-toxic stilbenes, pathogen growth inhibition and PCD.

The grapevine (Vitis vinifera) LysM receptor kinases VvLYK1-1 and VvLYK1-2 mediate chitooligosaccharide-triggered immunity.

Chitin, a major component of fungal cell walls, is a well-known pathogen-associated molecular pattern (PAMP) that triggers defense responses in several mammal and plant species. Here, we show that two chitooligosaccharides, chitin and chitosan, act as PAMPs in grapevine (Vitis vinifera) as they elicit immune signalling events, defense gene expression and resistance against fungal diseases. To identify their cognate receptors, the grapevine family of LysM receptor kinases (LysM-RKs) was annotated and their gene expression profiles were characterized. Phylogenetic analysis clearly distinguished three V. vinifera LysM-RKs (VvLYKs) located in the same clade as the Arabidopsis CHITIN ELICITOR RECEPTOR KINASE1 (AtCERK1), which mediates chitin-induced immune responses. The Arabidopsis mutant Atcerk1, impaired in chitin perception, was transformed with these three putative orthologous genes encoding VvLYK1-1, -2, or -3 to determine if they would complement the loss of AtCERK1 function. Our results provide evidence that VvLYK1-1 and VvLYK1-2, but not VvLYK1-3, functionally complement the Atcerk1 mutant by restoring chitooligosaccharide-induced MAPK activation and immune gene expression. Moreover, expression of VvLYK1-1 in Atcerk1 restored penetration resistance to the non-adapted grapevine powdery mildew (Erysiphe necator). On the whole, our results indicate that the grapevine VvLYK1-1 and VvLYK1-2 participate in chitin- and chitosan-triggered immunity and that VvLYK1-1 plays an important role in basal resistance against E. necator.

In Planta Functional Analysis and Subcellular Localization of the Oomycete Pathogen Plasmopara viticola Candidate RXLR Effector Repertoire.

Downy mildew is one of the most destructive diseases of grapevine, causing tremendous economic loss in the grape and wine industry. The disease agent Plasmopara viticola is an obligate biotrophic oomycete, from which over 100 candidate RXLR effectors have been identified. In this study, 83 candidate RXLR effector genes (PvRXLRs) were cloned from the P. viticola isolate "JL-7-2" genome. The results of the yeast signal sequence trap assay indicated that most of the candidate effectors are secretory proteins. The biological activities and subcellular localizations of all the 83 effectors were analyzed via a heterologous Agrobacterium-mediated Nicotiana benthamiana expression system. Results showed that 52 effectors could completely suppress cell death triggered by elicitin, 10 effectors could partially suppress cell death, 11 effectors were unable to suppress cell death, and 10 effectors themselves triggered cell death. Live-cell imaging showed that the majority of the effectors (76 of 83) could be observed with informative fluorescence signals in plant cells, among which 34 effectors were found to be targeted to both the nucleus and cytosol, 29 effectors were specifically localized in the nucleus, and 9 effectors were targeted to plant membrane system. Interestingly, three effectors PvRXLR61, 86 and 161 were targeted to chloroplasts, and one effector PvRXLR54 was dually targeted to chloroplasts and mitochondria. However, western blot analysis suggested that only PvRXLR86 carried a cleavable N-terminal transit peptide and underwent processing in planta. Many effectors have previously been predicted to target organelles, however, to the best of our knowledge, this is the first study to provide experimental evidence of oomycete effectors targeted to chloroplasts and mitochondria.

Combinatorial Regulation of Stilbene Synthase Genes by WRKY and MYB Transcription Factors in Grapevine (Vitis vinifera L.).

Stilbene synthase (STS) is the key enzyme leading to the biosynthesis of resveratrol. Recently we reported two R2R3-MYB transcription factor (TF) genes that regulate the stilbene biosynthetic pathway in grapevine: VviMYB14 and VviMYB15. These genes are strongly co-expressed with STS genes under a range of stress and developmental conditions, in agreement with the specific activation of STS promoters by these TFs. Genome-wide gene co-expression analysis using two separate transcriptome compendia based on microarray and RNA sequencing data revealed that WRKY TFs were the top TF family correlated with STS genes. On the basis of correlation frequency, four WRKY genes, namely VviWRKY03, VviWRKY24, VviWRKY43 and VviWRKY53, were further shortlisted and functionally validated. Expression analyses under both unstressed and stressed conditions, together with promoter-luciferase reporter assays, suggested different hierarchies for these TFs in the regulation of the stilbene biosynthetic pathway. In particular, VviWRKY24 seems to act as a singular effector in the activation of the VviSTS29 promoter, while VviWRKY03 acts through a combinatorial effect with VviMYB14, suggesting that these two regulators may interact at the protein level as previously reported in other species.

Genome sequence of Plasmopara viticola and insight into the pathogenic mechanism.

Plasmopara viticola causes downy mildew disease of grapevine which is one of the most devastating diseases of viticulture worldwide. Here we report a 101.3 Mb whole genome sequence of P. viticola isolate 'JL-7-2' obtained by a combination of Illumina and PacBio sequencing technologies. The P. viticola genome contains 17,014 putative protein-coding genes and has ~26% repetitive sequences. A total of 1,301 putative secreted proteins, including 100 putative RXLR effectors and 90 CRN effectors were identified in this genome. In the secretome, 261 potential pathogenicity genes and 95 carbohydrate-active enzymes were predicted. Transcriptional analysis revealed that most of the RXLR effectors, pathogenicity genes and carbohydrate-active enzymes were significantly up-regulated during infection. Comparative genomic analysis revealed that P. viticola evolved independently from the Arabidopsis downy mildew pathogen Hyaloperonospora arabidopsidis. The availability of the P. viticola genome provides a valuable resource not only for comparative genomic analysis and evolutionary studies among oomycetes, but also enhance our knowledge on the mechanism of interactions between this biotrophic pathogen and its host.

Structure and Function of the TIR Domain from the Grape NLR Protein RPV1.

The N-terminal Toll/interleukin-1 receptor/resistance protein (TIR) domain has been shown to be both necessary and sufficient for defense signaling in the model plants flax and Arabidopsis. In examples from these organisms, TIR domain self-association is required for signaling function, albeit through distinct interfaces. Here, we investigate these properties in the TIR domain containing resistance protein RPV1 from the wild grapevine Muscadinia rotundifolia. The RPV1 TIR domain, without additional flanking sequence present, is autoactive when transiently expressed in tobacco, demonstrating that the TIR domain alone is capable of cell-death signaling. We determined the crystal structure of the RPV1 TIR domain at 2.3 Å resolution. In the crystals, the RPV1 TIR domain forms a dimer, mediated predominantly through residues in the αA and αE helices ("AE" interface). This interface is shared with the interface discovered in the dimeric complex of the TIR domains from the Arabidopsis RPS4/RRS1 resistance protein pair. We show that surface-exposed residues in the AE interface that mediate the dimer interaction in the crystals are highly conserved among plant TIR domain-containing proteins. While we were unable to demonstrate self-association of the RPV1 TIR domain in solution or using yeast 2-hybrid, mutations of surface-exposed residues in the AE interface prevent the cell-death autoactive phenotype. In addition, mutation of residues known to be important in the cell-death signaling function of the flax L6 TIR domain were also shown to be required for RPV1 TIR domain mediated cell-death. Our data demonstrate that multiple TIR domain surfaces control the cell-death function of the RPV1 TIR domain and we suggest that the conserved AE interface may have a general function in TIR-NLR signaling.

Identification of two novel powdery mildew resistance loci, Ren6 and Ren7, from the wild Chinese grape species Vitis piasezkii.

BACKGROUND: Grapevine powdery mildew Erysiphe necator is a major fungal disease in all grape growing countries worldwide. Breeding for resistance to this disease is crucial to avoid extensive fungicide applications that are costly, labor intensive and may have detrimental effects on the environment. In the past decade, Chinese Vitis species have attracted attention from grape breeders because of their strong resistance to powdery mildew and their lack of negative fruit quality attributes that are often present in resistant North American species. In this study, we investigated powdery mildew resistance in multiple accessions of the Chinese species Vitis piasezkii that were collected during the 1980 Sino-American botanical expedition to the western Hubei province of China. RESULTS: A framework genetic map was developed using simple sequence repeat markers in 277 seedlings of an F1 mapping population arising from a cross of the powdery mildew susceptible Vitis vinifera selection F2-35 and a resistant accession of V. piasezkii DVIT2027. Quantitative trait locus analyses identified two major powdery mildew resistance loci on chromosome 9 (Ren6) and chromosome 19 (Ren7) explaining 74.8 % of the cumulative phenotypic variation. The quantitative trait locus analysis for each locus, in the absence of the other, explained 95.4 % phenotypic variation for Ren6, while Ren7 accounted for 71.9 % of the phenotypic variation. Screening of an additional 259 seedlings of the F1 population and 910 seedlings from four pseudo-backcross populations with SSR markers defined regions of 22 kb and 330 kb for Ren6 and Ren7 in the V. vinifera PN40024 (12X) genome sequence, respectively. Both R loci operate post-penetration through the induction of programmed cell death, but vary significantly in the speed of response and degree of resistance; Ren6 confers complete resistance whereas Ren7 confers partial resistance to the disease with reduced colony size. A comparison of the kinetics of induction of powdery mildew resistance mediated by Ren6, Ren7 and the Run1 locus from Muscadinia rotundifolia, indicated that the speed and strength of resistance conferred by Ren6 is greater than that of Run1 which, in turn, is superior to that conferred by Ren7. CONCLUSIONS: This is the first report of mapping powdery mildew resistance in the Chinese species V. piasezkii. Two distinct powdery mildew R loci designated Ren6 and Ren7 were found in multiple accessions of this Chinese grape species. Their location on different chromosomes to previously reported powdery mildew resistance R loci offers the potential for grape breeders to combine these R genes with existing powdery mildew R loci to produce grape germplasm with more durable resistance against this rapidly evolving fungal pathogen.

Phylogenetic Relationships of Pseudomonas syringae pv. syringae Isolates Associated with Bacterial Inflorescence Rot in Grapevine.

Pseudomonas syringae pv. syringae causes extensive yield losses in wine-grape production in some Australian cool-climate vineyards. Putative P. syringae pv. syringae isolates from infected grapevines within a range of vineyards were genotyped using RNA polymerase ß-subunit (rpoB) and multilocus sequence typing (MLST) using primers for glyceraldehyde-3-phosphate dehydrogenase (gapA), citrate synthase (gltA), DNA gyrase B (gyrB), and σ factor 70 (rpoD). The isolates were also evaluated for pathogenicity by inoculation of detached grapevine leaves. The isolates were grouped by MLST data into two well-supported clades, each containing a mixture of pathogenic and nonpathogenic grapevine isolates, indicating that P. syringae pv. syringae in Australian vineyards is genetically diverse. Each clade also contained P. syringae pv. syringae from nongrape hosts pathogenic to grapevine, demonstrating a lack of host specificity and possible potential for cross-infection of grape and other horticultural crops. Furthermore, the isolation of pathogenic P. syringae pv. syringae isolates from grapevine sucker shoots suggests that sucker shoots may allow overwintering of the pathogen. The vineyard quarantine status of P. syringae pv. syringae may need to be reconsidered, due to its easy dispersal through pruning equipment.

Current understanding of grapevine defense mechanisms against the biotrophic fungus (Erysiphe necator), the causal agent of powdery mildew disease.

The most economically important disease of cultivated grapevines worldwide is powdery mildew (PM) caused by the ascomycete fungus Erysiphe necator. The majority of grapevine cultivars used for wine, table grape, and dried fruit production are derived from the Eurasian grape species Vitis vinifera because of its superior aroma and flavor characteristics. However, this species has little genetic resistance against E. necator meaning that grape production is highly dependent on the frequent use of fungicides. The integration of effective genetic resistance into cultivated grapevines would lead to significant financial and environmental benefits and represents a major challenge for viticultural industries and researchers worldwide. This review will outline the strategies being used to increase our understanding of the molecular basis of V. vinifera susceptibility to this fungal pathogen. It will summarize our current knowledge of different resistance loci/genes that have evolved in wild grapevine species to restrict PM infection and assess the potential application of these defense genes in the generation of PM-resistant grapevine germplasm. Finally, it addresses future research priorities which will be important in the rapid identification, evaluation, and deployment of new PM resistance genes which are capable of conferring effective and durable resistance in the vineyard.

Strategies for RUN1 Deployment Using RUN2 and REN2 to Manage Grapevine Powdery Mildew Informed by Studies of Race Specificity.

The Toll/interleukin-1 receptor nucleotide-binding site leucine-rich repeat gene, "resistance to Uncinula necator 1" (RUN1), from Vitis rotundifolia was recently identified and confirmed to confer resistance to the grapevine powdery mildew fungus Erysiphe necator (syn. U. necator) in transgenic V. vinifera cultivars. However, sporulating powdery mildew colonies and cleistothecia of the heterothallic pathogen have been found on introgression lines containing the RUN1 locus growing in New York (NY). Two E. necator isolates collected from RUN1 vines were designated NY1-131 and NY1-137 and were used in this study to inform a strategy for durable RUN1 deployment. In order to achieve this, fitness parameters of NY1-131 and NY1-137 were quantified relative to powdery mildew isolates collected from V. rotundifolia and V. vinifera on vines containing alleles of the powdery mildew resistance genes RUN1, RUN2, or REN2. The results clearly demonstrate the race specificity of RUN1, RUN2, and REN2 resistance alleles, all of which exhibit programmed cell death (PCD)-mediated resistance. The NY1 isolates investigated were found to have an intermediate virulence on RUN1 vines, although this may be allele specific, while the Musc4 isolate collected from V. rotundifolia was virulent on all RUN1 vines. Another powdery mildew resistance locus, RUN2, was previously mapped in different V. rotundifolia genotypes, and two alleles (RUN2.1 and RUN2.2) were identified. The RUN2.1 allele was found to provide PCD-mediated resistance to both an NY1 isolate and Musc4. Importantly, REN2 vines were resistant to the NY1 isolates and RUN1REN2 vines combining both genes displayed additional resistance. Based on these results, RUN1-mediated resistance in grapevine may be enhanced by pyramiding with RUN2.1 or REN2; however, naturally occurring isolates in North America display some virulence on vines with these resistance genes. The characterization of additional resistance sources is needed to identify resistance gene combinations that will further enhance durability. For the resistance gene combinations currently available, we recommend using complementary management strategies, including fungicide application, to reduce populations of virulent isolates.

Simple quantification of in planta fungal biomass.

An accurate assessment of the disease resistance status of plants to fungal pathogens is an essential requirement for the development of resistant crop plants. Many disease resistance phenotypes are partial rather than obvious immunity and are frequently scored using subjective qualitative estimates of pathogen development or plant disease symptoms. Here we report a method for the accurate comparison of total fungal biomass in plant tissues. This method, called the WAC assay, is based upon the specific binding of the plant lectin wheat germ agglutinin to fungal chitin. The assay is simple, high-throughput, and sensitive enough to discriminate between single Puccinia graminis f.sp tritici infection sites on a wheat leaf segment. It greatly lends itself to replication as large volumes of tissue can be pooled from independent experiments and assayed to provide truly representative quantification, or, alternatively, fungal growth on a single, small leaf segment can be quantified. In addition, as the assay is based upon a microscopic technique, pathogen infection sites can also be examined at high magnification prior to quantification if desired and average infection site areas are determined. Previously, we have demonstrated the application of the WAC assay for quantifying the growth of several different pathogen species in both glasshouse grown material and large-scale field plots. Details of this method are provided within.

The R2R3-MYB transcription factors MYB14 and MYB15 regulate stilbene biosynthesis in Vitis vinifera.

Plant stilbenes are phytoalexins that accumulate in a small number of plant species, including grapevine (Vitis vinifera), in response to biotic and abiotic stresses and have been implicated in many beneficial effects on human health. In particular, resveratrol, the basic unit of all other complex stilbenes, has received widespread attention because of its cardio-protective, anticarcinogenic, and antioxidant properties. Although stilbene synthases (STSs), the key enzymes responsible for resveratrol biosynthesis, have been isolated and characterized from several plant species, the transcriptional regulation underlying stilbene biosynthesis is unknown. Here, we report the identification and functional characterization of two R2R3-MYB-type transcription factors (TFs) from grapevine, which regulate the stilbene biosynthetic pathway. These TFs, designated MYB14 and MYB15, strongly coexpress with STS genes, both in leaf tissues under biotic and abiotic stress and in the skin and seed of healthy developing berries during maturation. In transient gene reporter assays, MYB14 and MYB15 were demonstrated to specifically activate the promoters of STS genes, and the ectopic expression of MYB15 in grapevine hairy roots resulted in increased STS expression and in the accumulation of glycosylated stilbenes in planta. These results demonstrate the involvement of MYB14 and MYB15 in the transcriptional regulation of stilbene biosynthesis in grapevine.

Genetic dissection of a TIR-NB-LRR locus from the wild North American grapevine species Muscadinia rotundifolia identifies paralogous genes conferring resistance to major fungal and oomycete pathogens in cultivated grapevine.

The most economically important diseases of grapevine cultivation worldwide are caused by the fungal pathogen powdery mildew (Erysiphe necator syn. Uncinula necator) and the oomycete pathogen downy mildew (Plasmopara viticola). Currently, grapegrowers rely heavily on the use of agrochemicals to minimize the potentially devastating impact of these pathogens on grape yield and quality. The wild North American grapevine species Muscadinia rotundifolia was recognized as early as 1889 to be resistant to both powdery and downy mildew. We have now mapped resistance to these two mildew pathogens in M. rotundifolia to a single locus on chromosome 12 that contains a family of seven TIR-NB-LRR genes. We further demonstrate that two highly homologous (86% amino acid identity) members of this gene family confer strong resistance to these unrelated pathogens following genetic transformation into susceptible Vitis vinifera winegrape cultivars. These two genes, designated resistance to Uncinula necator (MrRUN1) and resistance to Plasmopara viticola (MrRPV1) are the first resistance genes to be cloned from a grapevine species. Both MrRUN1 and MrRPV1 were found to confer resistance to multiple powdery and downy mildew isolates from France, North America and Australia; however, a single powdery mildew isolate collected from the south-eastern region of North America, to which M. rotundifolia is native, was capable of breaking MrRUN1-mediated resistance. Comparisons of gene organization and coding sequences between M. rotundifolia and the cultivated grapevine V. vinifera at the MrRUN1/MrRPV1 locus revealed a high level of synteny, suggesting that the TIR-NB-LRR genes at this locus share a common ancestor.

A simple method for comparing fungal biomass in infected plant tissues.

Plant phenotypes resistant and susceptible to fungal pathogens are usually scored using qualitative, subjective methods that are based upon disease symptoms or by an estimation of the amount of visible fungal growth. Given that plant resistance genes often confer partial resistance to fungal pathogens, a simple, sensitive, nonsubjective quantitative method for measuring pathogen growth would be highly advantageous. This report describes an in planta quantitative assay for fungal biomass based upon detection of chitin using wheat germ agglutinin conjugated to a fluorophore. Using this assay, the growth of wheat rust pathogens on wheat was assayed and the additivity of several adult plant and seedling resistance genes to Puccinia striiformis, P. graminis, and P. triticina was assayed on both glasshouse- and field-grown material. The assay can discriminate between individual rust pustules on a leaf segment or, alternatively, compare fungal growth on field plots. The quantification of Erysiphe necator (powdery mildew) growth on Vitis vinifera (grapevine) is also demonstrated, with resistant and susceptible cultivars readily distinguished. Given that chitin is a major cell wall component of many plant fungal pathogens, this robust assay will enable simple and accurate measurement of biomass accumulation in many plant-fungus interactions.