“The PLCP gene family of grapevine (Vitis vinifera L.): characterization and differential expression in response to Plasmopara Viticola”

Abstract Background Papain-like cysteine proteases (PLCPs), a large group of cysteine proteases, are structurally related to papain. The members belonging to PLCPs family contribute to plant immunity, senescence, and defense responses in plants. The PLCP gene family has been identified in Arabidopsis, rice, soybean, and cotton. However, no systematic analysis of PLCP genes has been undertaken in grapevine. Since Plasmopara viticola as a destructive pathogen could affect immunity of grapes in the field, we considered that the members belonged to PLCPs family could play a crucial role in defensive mechanisms or programmed cell death. We aimed to evaluate the role of PLCPs in 2 different varieties of grapevines and compared the changes of their expressions with the transcriptional data in response to P. viticola. Results In this study, 23 grapevine PLCP (VvPLCP) genes were identified by comprehensive bioinformatics analysis. Subsequently, the chromosomal localizations, gene structure, conserved domains, phylogenetic relationship, gene duplication, and cis-acting elements were analyzed. Numerous cis-acting elements related to plant development, hormone, and stress responses were identified in the promoter of the VvPLCP genes. Phylogenetic analysis grouped the VvPLCP genes into nine subgroups. The transcription of VvPLCP in different inoculation time points and varieties indicated that VvPLCP may have vital functions in grapevine defense against Plasmopara viticola. According to transcriptome data and qPCR analysis, we observed the increasing expression levels of VvRD21–1 at 72 h after inoculation in resistant variety, inferring that it was related to grape downy mildew resistance. Meanwhile, 3 genes including VvXBCP1, VvSAG12–1, and VvALP1 showed higher expression at 24 h after pathogen inoculation in the susceptible variety and might be related to the downy mildew phenotype. We nominated these four genes to function during hypersensitive response (HR) process, inferring that these genes could be associated with downy mildew resistance in grapes. Conclusions Our results provide the reference for functional studies of PLCP gene family, and highlight its functions in grapevine defense against P. viticola. The results help us to better understand the complexity of the PLCP gene family in plant immunity and provide valuable information for future functional characterization of specific genes in grapevine.

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Structural and molecular characterization of the Rpv3 locus towards the development of KASP markers for downy mildew resistance in grapevine (Vitis spp.)

10.1101/2021.02.25.432814 ◽

2021 ◽

Author(s):

Andriele Wairich ◽

Jaiana Malabarba ◽

Vanessa Buffon ◽

Diogo Denardi Porto ◽

Roberto Togawa ◽

...

Keyword(s):

Candidate Genes ◽

Downy Mildew ◽

Plasmopara Viticola ◽

Functional Enrichment ◽

Downy Mildew Resistance ◽

Differential Analysis ◽

Mildew Resistance ◽

Grapevine Cultivar ◽

Genomic Characterization

AbstractPlasmopara viticola is the oomycete that causes downy mildew in grapevine. Varying levels of resistance to P. viticola across grape cultivars allowed quantitative trait loci to be identified. The Rpv3 locus is located at chromosome 18, in a region enriched in TIR-NBS-LRR genes, and the phenotype associated is a high hypersensitive response. In this work, we aimed to identify candidate genes associated with resistance to downy mildew on the Rpv3 locus and to evaluate their transcriptional profiles in a susceptible and a resistant grapevine cultivar after challenging with P. viticola. Candidate genes were identified by representational differential analysis and also by functional enrichment tests. Many predicted genes associated with resistance to diseases were found at the Rpv3 locus. In total, seventeen genes were evaluated by RT-qPCR. Differences in the steady-state expression of these genes were observed between the two cultivars. Four genes were found to be expressed only in Villard Blanc, suggesting their association to the hypersensitivity reaction. Concerning marker assisted-selection for downy mildew resistance, we show the efficient use of a haplotype of SSR markers. Furthermore, based on Rpv3-located SNPs between grapevine cultivars contrasting in downy mildew resistance, we developed and tested forty-one new markers for assisted selection. After genotypic and phenotypic evaluations on segregant populations, two markers, Rpv3_15 and Rpv3_33, were considered efficient for downy mildew resistance identification. This study constitutes an in-depth genomic characterization of the Rpv3 locus, confirms its involvement in resistance against P. viticola infection and presents promising biotechnological tools for the selection of young resistant individuals.

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Variation Within and Between Vitis spp. for Foliar Resistance to the Downy Mildew Pathogen Plasmopara viticola

Plant Disease ◽

10.1094/pdis-92-11-1577 ◽

2008 ◽

Vol 92 (11) ◽

pp. 1577-1584 ◽

Cited By ~ 48

Author(s):

Lance Cadle-Davidson

Keyword(s):

Downy Mildew ◽

Control Strategies ◽

Natural Infection ◽

Leaf Age ◽

Plasmopara Viticola ◽

Downy Mildew Resistance ◽

Mildew Resistance ◽

Detached Leaf ◽

Foliar Resistance ◽

Field Assay

To complement existing control strategies, grape growers in humid climates desire cultivars with resistance to downy mildew caused by Plasmopara viticola. Numerous disease resistance screens of diverse Vitis germplasm have been conducted previously to identify downy mildew resistance; however, ratings of named cultivars were inconsistent and identities of resistant individuals in wild species were not typically provided. Inconsistencies among previous studies could be due to race-specific resistance. In the current study, controlled inoculations of two single isolates onto two leaf ages of 883 Vitis accessions were used and these results compared with natural infection in a fivefold replicated vineyard of 80 Vitis accessions in 2006 and 2007. Of the accessions rated in both assays, 16.2% were resistant to a single isolate but susceptible in the vineyard. Otherwise, there was good correlation of ratings between the field assay and the rating of older leaves (r = 0.62 to 0.71). Five accessions from Vitis cinerea, V. labrusca, and Vitis × champinii averaged zero severity in both vineyard years, yet some individuals of V. cinerea and V. labrusca were moderately or highly susceptible in the field. Similarly, although significant differences in mean severity separated V. vinifera, Vitis hybrid, V. riparia, and V. labrusca for single-isolate inoculations (from susceptible to resistant), notable intraspecies variation was identified for all well-represented species. Resistant individuals were identified in most species with the prominent exceptions of V. vinifera and V. acerifolia. Single-isolate, detached-leaf resistance ratings in 2006 corresponded well (94.6%) to 2007 ratings using a separate isolate collected from the same vineyard. Categorizing the ratings for this and previous studies, ratings infrequently corresponded among previous studies (31.9%) as well as between previous studies and the current single-isolate (34.9%) or vineyard (46.4%) ratings. These results highlight important factors for downy mildew resistance screens: leaf age, pathogen genotype, and host species and accession. The results further underscore the importance to breeders of uniform testing in multiple environments.

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Inheritance of Downy Mildew Resistance in Table Grapes

Journal of the American Society for Horticultural Science ◽

10.21273/jashs.124.3.262 ◽

1999 ◽

Vol 124 (3) ◽

pp. 262-267 ◽

Cited By ~ 8

Author(s):

Maurus V. Brown ◽

James N. Moore ◽

Ronald W. McNew ◽

Patrick Fenn

Keyword(s):

Downy Mildew ◽

Combining Ability ◽

Plasmopara Viticola ◽

Downy Mildew Resistance ◽

Resistant Parent ◽

Mildew Resistance ◽

Table Grapes ◽

Additive Gene ◽

Foliar Tissue ◽

Different Levels

A study was conducted to determine how resistance to downy mildew [Plasmopara viticola (Bert. & Curt.) Berl. & de Toni] is inherited in germplasm (Vitis vinifera L., V. labrusca L., V. rupestris Scheele, and V. riparia Michx.) used for breeding table grapes. Crosses, including reciprocals, among parents possessing different levels of downy mildew resistance were evaluated in 1994 and 1995. The proportion of foliar tissue with sporulation, chlorosis, or necrosis was used to measure resistance. All genotypes were rated for these characters on two separate dates in 1994 and 1995. Hypersensitive flecking was also evaluated in the 1995 seedlings to determine its relationship with downy mildew resistance. Crosses with at least one resistant parent had a larger number of resistant offspring than crosses between two susceptible parents. General combining ability (GCA) effects were highly significant for 1994 and 1995. Specific combining ability effects were significant, but were relatively small compared to GCA, suggesting additive gene action was a primary influence on downy mildew resistance. Heritability estimates for sporulation, chlorosis, and necrosis were the highest at the second rating in 1994 (0.88, 0.74, and 0.57, respectively) and 1995 (0.50, 0.60, and 0.60, respectively). Reciprocal crosses indicated that maternal inheritance did not influence downy mildew resistance. A small percentage of progeny with hypersensitive flecking were identified from the germplasm. Seedlings with the flecking characteristic tended to have lower sporulation, chlorosis, and necrosis ratings earlier in the growing season.

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An Extended Leaf Disc Test for Virulence Assessment in Plasmopara Viticola and Detection of Downy Mildew Resistance in Vitis

Journal of Plant Pathology & Microbiology ◽

10.4172/2157-7471.1000353 ◽

2016 ◽

Vol 07 (05) ◽

Cited By ~ 2

Author(s):

Javier Gómez-Zeledón ◽

Markus Kaiser

Keyword(s):

Downy Mildew ◽

Leaf Disc ◽

Plasmopara Viticola ◽

Downy Mildew Resistance ◽

Mildew Resistance

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SCREENING TECHNIQUES FOR DOWNY MILDEW RESISTANCE IN GHERKIN CUCUMBERS

International Journal of Geomate ◽

10.21660/2017.40.2603 ◽

2017 ◽

Vol 13 (40) ◽

Author(s):

Piyavadee Charoenwattana

Keyword(s):

Downy Mildew ◽

Downy Mildew Resistance ◽

Mildew Resistance ◽

Screening Techniques

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Investigation of long non-coding RNAs as regulatory players of grapevine response to powdery and downy mildew infection

BMC Plant Biology ◽

10.1186/s12870-021-03059-6 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Garima Bhatia ◽

Santosh K. Upadhyay ◽

Anuradha Upadhyay ◽

Kashmir Singh

Keyword(s):

Downy Mildew ◽

Plasmopara Viticola ◽

Defense Responses ◽

Protein Coding ◽

Functional Roles ◽

Real Time Quantitative Pcr ◽

Transcriptional Reprogramming ◽

Sequencing Technologies ◽

Non Coding Rnas ◽

Fungal Phytopathogens

Abstract Background Long non-coding RNAs (lncRNAs) are regulatory transcripts of length > 200 nt. Owing to the rapidly progressing RNA-sequencing technologies, lncRNAs are emerging as considerable nodes in the plant antifungal defense networks. Therefore, we investigated their role in Vitis vinifera (grapevine) in response to obligate biotrophic fungal phytopathogens, Erysiphe necator (powdery mildew, PM) and Plasmopara viticola (downy mildew, DM), which impose huge agro-economic burden on grape-growers worldwide. Results Using computational approach based on RNA-seq data, 71 PM- and 83 DM-responsive V. vinifera lncRNAs were identified and comprehensively examined for their putative functional roles in plant defense response. V. vinifera protein coding sequences (CDS) were also profiled based on expression levels, and 1037 PM-responsive and 670 DM-responsive CDS were identified. Next, co-expression analysis-based functional annotation revealed their association with gene ontology (GO) terms for ‘response to stress’, ‘response to biotic stimulus’, ‘immune system process’, etc. Further investigation based on analysis of domains, enzyme classification, pathways enrichment, transcription factors (TFs), interactions with microRNAs (miRNAs), and real-time quantitative PCR of lncRNAs and co-expressing CDS pairs suggested their involvement in modulation of basal and specific defense responses such as: Ca2+-dependent signaling, cell wall reinforcement, reactive oxygen species metabolism, pathogenesis related proteins accumulation, phytohormonal signal transduction, and secondary metabolism. Conclusions Overall, the identified lncRNAs provide insights into the underlying intricacy of grapevine transcriptional reprogramming/post-transcriptional regulation to delay or seize the living cell-dependent pathogen growth. Therefore, in addition to defense-responsive genes such as TFs, the identified lncRNAs can be further examined and leveraged to candidates for biotechnological improvement/breeding to enhance fungal stress resistance in this susceptible fruit crop of economic and nutritional importance.

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High resolution mapping and candidate gene identification of downy mildew race 16 resistance in spinach

BMC Genomics ◽

10.1186/s12864-021-07788-8 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Gehendra Bhattarai ◽

Wei Yang ◽

Ainong Shi ◽

Chunda Feng ◽

Braham Dhillon ◽

...

Keyword(s):

Candidate Genes ◽

Downy Mildew ◽

Association Analysis ◽

Spinacia Oleracea ◽

Genotyping By Sequencing ◽

Significant Snps ◽

Snp Markers ◽

Resistance Locus ◽

Downy Mildew Resistance ◽

Mildew Resistance

Abstract Background Downy mildew, the most devastating disease of spinach (Spinacia oleracea L.), is caused by the oomycete Peronospora effusa [=P. farinosa f. sp. spinaciae]. The P. effusa shows race specificities to the resistant host and comprises 19 reported races and many novel isolates. Sixteen new P. effusa races were identified during the past three decades, and the new pathogen races are continually overcoming the genetic resistances used in commercial cultivars. A spinach breeding population derived from the cross between cultivars Whale and Lazio was inoculated with P. effusa race 16 in an environment-controlled facility; disease response was recorded and genotyped using genotyping by sequencing (GBS). The main objective of this study was to identify resistance-associated single nucleotide polymorphism (SNP) markers from the cultivar Whale against the P. effusa race 16. Results Association analysis conducted using GBS markers identified six significant SNPs (S3_658,306, S3_692697, S3_1050601, S3_1227787, S3_1227802, S3_1231197). The downy mildew resistance locus from cultivar Whale was mapped to a 0.57 Mb region on chromosome 3, including four disease resistance candidate genes (Spo12736, Spo12784, Spo12908, and Spo12821) within 2.69–11.28 Kb of the peak SNP. Conclusions Genomewide association analysis approach was used to map the P. effusa race 16 resistance loci and identify associated SNP markers and the candidate genes. The results from this study could be valuable in understanding the genetic basis of downy mildew resistance, and the SNP marker will be useful in spinach breeding to select resistant lines.

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Mutants of downy mildew resistance in Lactuca sativa (lettuce).

Genetics ◽

10.1093/genetics/137.3.867 ◽

1994 ◽

Vol 137 (3) ◽

pp. 867-874

Author(s):

P A Okubara ◽

P A Anderson ◽

O E Ochoa ◽

R W Michelmore

Keyword(s):

Molecular Marker ◽

Downy Mildew ◽

Genomic Dna ◽

Spontaneous Mutation ◽

Multigene Families ◽

Downy Mildew Resistance ◽

Bremia Lactucae ◽

Wild Type ◽

Mildew Resistance ◽

Recessive Mutations

Abstract As part of our investigation of disease resistance in lettuce, we generated mutants that have lost resistance to Bremia lactucae, the casual fungus of downy mildew. Using a rapid and reliable screen, we identified 16 distinct mutants of Latuca sativa that have lost activity of one of four different downy mildew resistance genes (Dm). In all mutants, only a single Dm specificity was affected. Genetic analysis indicated that the lesions segregated as single, recessive mutations at the Dm loci. Dm3 was inactivated in nine of the mutants. One of five Dm 1 mutants was selected from a population of untreated seeds and therefore carried a spontaneous mutation. All other Dm1, Dm3, Dm5/8 and Dm7 mutants were derived from gamma- or fast neutron-irradiated seed. In two separate Dm 1 mutants and in each of the eight Dm3 mutants analyzed, at least one closely linked molecular marker was absent. Also, high molecular weight genomic DNA fragments that hybridized to a tightly linked molecular marker in wild type were either missing entirely or were truncated in two of the Dm3 mutants, providing additional evidence that deletions had occurred in these mutants. Absence of mutations at loci epistatic to the Dm genes suggested that such loci were either members of multigene families, were critical for plant survival, or encoded components of duplicated pathways for resistance; alternatively, the genes determining downy mildew resistance might be limited to the Dm loci.

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