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

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.

Lines resistant to downy mildew in the sunflower genetic collection at VIR

Background. Downy mildew (DM) caused by the fungus Plasmopara halstedii (Farl) Berl. & De Toni) is one of the most harmful diseases of sunflower (Helianthus annuus L.). Due to the pathogen’s attacks, annual seed harvest losses range between 30% and 70%. Lines resistant to new races of the pathogen should be obtained for the development of commercial sunflower hybrids.Materials and methods. Downy mildew resistance of 323 lines and 10 cultivars from the sunflower collection was assessed in the field at the Kuban Experiment Station of VIR in 2017–2018. Line VIR 845, susceptible during all years of observations, was used as a control. Resistance genes were identified by means of the molecular analysis using diagnostic markers of the Plarg, Pl6 and Pl8 genes that confer resistance to many known P. halstedii races.Results. The founder varieties of VIR’s lines were susceptible to a varying extent. Thirty-nine lines were resistant in 2016 and 2018; among those, 36 lines were susceptible in 2017. Presumably, a more virulent P. halstedii race became widespread in 2017, compared to the races that prevailed in 2016 and 2018, so the genes that determined resistance in 2016 and 2018 turned out to be ineffective. Lines ТА 716-18, VIR 768, and VIR 800, having originated from interspecific hybrids, exhibited absence of pathogenic damage during 3 years of the trials. Molecular markers of the Plarg, Pl6 and Pl8 genes were detected in most lines that demonstrated resistance in 2016 and 2018. There were no markers in lines VIR 768 and VIR 800, whereas in ТА 716-18 the markers of Plarg and Pl8 were present.Conclusion. As a result of the long-term studies, a trait-specific genetic collection was established for sunflower. It comprises genotyped lines with various effective DM resistance genes. Lines ТА 716-18, VIR 768 and VIR 800 appeared highly resistant to the pathogen and probably possess new resistance genes/alleles introgressed from wild species.

Molecular Breeding Strategy and Challenges Towards Improvement of Downy Mildew Resistance in Cauliflower (Brassica oleracea var. botrytis L.)

Cauliflower (Brassica oleracea var. botrytis L.) is one of the important, nutritious and healthy vegetable crops grown and consumed worldwide. But its production is constrained by several destructive fungal diseases and most importantly, downy mildew leading to severe yield and quality losses. For sustainable cauliflower production, developing resistant varieties/hybrids with durable resistance against broad-spectrum of pathogens is the best strategy for a long term and reliable solution. Identification of novel resistant resources, knowledge of the genetics of resistance, mapping and cloning of resistance QTLs and identification of candidate genes would facilitate molecular breeding for disease resistance in cauliflower. Advent of next-generation sequencing technologies (NGS) and publishing of draft genome sequence of cauliflower has opened the flood gate for new possibilities to develop enormous amount of genomic resources leading to mapping and cloning of resistance QTLs. In cauliflower, several molecular breeding approaches such as QTL mapping, marker-assisted backcrossing, gene pyramiding have been carried out to develop new resistant cultivars. Marker-assisted selection (MAS) would be beneficial in improving the precision in the selection of improved cultivars against multiple pathogens. This comprehensive review emphasizes the fascinating recent advances made in the application of molecular breeding approach for resistance against an important pathogen; Downy Mildew (Hyaloperonospora parasitica) affecting cauliflower and Brassica oleracea crops and highlights the QTLs identified imparting resistance against this pathogen. We have also emphasized the critical research areas as future perspectives to bridge the gap between availability of genomic resources and its utility in identifying resistance genes/QTLs to breed downy mildew resistant cultivars. Additionally, we have also discussed the challenges and the way forward to realize the full potential of molecular breeding for downy mildew resistance by integrating marker technology with conventional breeding in the post-genomics era. All this information will undoubtedly provide new insights to the researchers in formulating future breeding strategies in cauliflower to develop durable resistant cultivars against the major pathogens in general and downy mildew in particular.

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

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.

CRISPR/Cas9-mediated mutagenesis of sweet basil candidate susceptibility gene ObDMR6 enhances downy mildew resistance

Sweet basil (Ocimum basilicum) is an economically important allotetraploid (2n = 4x = 48) herb whose global production is threatened by downy mildew disease caused by the obligate biotrophic oomycete, Peronospora belbahrii. Generation of disease resistant cultivars by mutagenesis of susceptibility (S) genes via CRISPR/Cas9 is currently one of the most promising strategies to maintain favored traits while improving disease resistance. Previous studies have identified Arabidopsis DMR6 (Downy Mildew Resistance 6) as an S gene required for pathogenesis of the downy mildew-causing oomycete pathogen Hyaloperonospora arabidopsidis. In this study, a sweet basil homolog of DMR6, designated ObDMR6, was identified in the popular sweet basil cultivar Genoveser and found to exist with a high copy number in the genome with polymorphisms among the variants. Two CRISPR/Cas9 constructs expressing one or two single guide RNAs (sgRNAs) targeting the conserved regions of ObDMR6 variants were generated and used to transform Genoveser via Agrobacterium-mediated transformation. 56 T0 lines were generated, and mutations of ObDMR6 were detected by analyzing the Sanger sequencing chromatograms of an ObDMR6 fragment using the Interference of CRISPR Edits (ICE) software. Among 54 lines containing mutations in the targeted sites, 13 had an indel percentage greater than 96% suggesting a near-complete knockout (KO) of ObDMR6. Three representative transgene-free lines with near-complete KO of ObDMR6 determined by ICE were identified in the T1 segregating populations derived from three independent T0 lines. The mutations were further confirmed using amplicon deep sequencing. Disease assays conducted on T2 seedlings of the above T1 lines showed a reduction in production of sporangia by 61–68% compared to the wild-type plants and 69–93% reduction in relative pathogen biomass determined by quantitative PCR (qPCR). This study not only has generated transgene-free sweet basil varieties with improved downy mildew resistance, but also contributed to our understanding of the molecular interactions of sweet basil-P. belbahrii.

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

Plasmopara 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.