Kinase Inhibitor VvBKI1 Interacts with Ascorbate Peroxidase VvAPX1 Promoting Plant Resistance to Oomycetes.

Downy mildew caused by oomycete pathogen Plasmopara viticola is a devastating disease of grapevine. P. viticola secretes an array of RXLR effectors to enhance virulence. One of these effectors, PvRXLR131, has been reported to interact with grape (Vitis vinifera) BRI1 kinase inhibitor (VvBKI1). BKI1 is conserved in Nicotiana benthamiana and Arabidopsis thaliana. However, the role of VvBKI1 in plant immunity is unknown. Here, we found transient expression of VvBKI1 in grapevine and N. benthamiana increased its resistance to P. viticola and Phytophthora capsici, respectively. Furthermore, ectopic expression of VvBKI1 in Arabidopsis can increase its resistance to downy mildew caused by Hyaloperonospora arabidopsidis. Further experiments revealed that VvBKI1 interacts with a cytoplasmic ascorbate peroxidase, VvAPX1, an ROS-scavenging protein. Transient expression of VvAPX1 in grape and N. benthamiana promoted its resistance against P. viticola, and P. capsici. Moreover, VvAPX1 transgenic Arabidopsis is more resistant to H. arabidopsidis. Furthermore, both VvBKI1 and VvAPX1 transgenic Arabidopsis showed an elevated ascorbate peroxidase activity and enhanced disease resistance. In summary, our findings suggest a positive correlation between APX activity and resistance to oomycetes and that this regulatory network is conserved in V. vinifera, N. benthamiana, and A. thaliana.

Genome sequence and comparative analysis of fungal antagonistic strain Bacillus velezensis LJBV19.

Bacillus species as fungal antagonistic agents have been widely used in the agriculture and considered as safe products for the management of plant pathogens. In this study, we reported the whole genome sequence of strain LJBV19 isolated from grapevine rhizosphere soil. Strain LJBV19 was identified as Bacillus velezensis through morphological, physicochemical, molecular analysis and genome comparison. Bacillus velezensis LJBV19 had a significant inhibitory effect on the growth of Magnaporthe oryzae with an inhibition ratio up to 75.55% and showed broad spectrum of activity against fungal phytopathogens. The 3,973,013-bp circular chromosome with an average GC content of 46.5% consisted of 3993 open reading frames (ORFs), and 3308 ORFs were classified into 19 cluster of orthologous groups of proteins (COG) categories. Genes related to cell wall degrading enzymes were predicted by Carbohydrate-Active enZYmes (CAZy) database and validated at the metabolic level, producing 0.53 ± 0.00 U/mL cellulose, 0.14 ± 0.01 U/mL chitinase, and 0.11 ± 0.01 U/mL chitosanase. Genome comparison confirmed the taxonomic position of LJBV19, conserved genomic structure, and genetic homogeneity. Moreover, 13 gene clusters for biosynthesis of secondary metabolites in LJBV19 genome were identified and two unique clusters (clusters 2 and 12) shown to direct an unknown compound were only present in strain LJBV19. In general, our results will provide insights into the antifungal mechanisms of Bacillus velezensis LJBV19 and further application of the strain.

Grapevine VaRPP13 protein enhances oomycetes resistance by activating SA signal pathway.

KEY MESSAGE: Expression of the VaRPP13 in Arabidopsis and tobacco enhanced resistance to oomycete pathogens, and this enhancement is closely related to the activation of salicylic acid (SA) signaling pathway. Resistance (R) genes, which usually contain a nucleotide-binding site and a leucine-rich repeat (NBS-LRR) domain, play crucial roles in disease resistance. In this study, we cloned a CC-NBS-LRR gene VaRPP13 from Vitis amurensis 'Shuang Hong' grapevine, and investigated its function on disease resistance. VaRPP13 expression was induced by Plasmopara viticola, an oomycetes pathogen causing downy mildew disease in grapevine. Heterologous expression VaRPP13 could also enhance resistance to Hyaloperonospora arabidopsidis in Arabidopsis thaliana and Phytophthora capsici in Nicotiana benthamiana, both oomycete pathogens. Further study indicated that VaRPP13 could enhance the expression of genes in SA signal pathway, while exogenous SA could also induce the expression of VaRPP13. In conclusion, our studies demonstrated that VaRPP13 contributes to a broad-spectrum resistance to oomycetes via activating SA signaling pathway.

Regulated deficit irrigation: an effective way to solve the shortage of agricultural water for horticulture.

The deficient agricultural water caused by water shortage is a crucial limiting factor of horticultural production. Among many agricultural water-saving technologies, regulated deficit irrigation (RDI) has been proven to be one of the effective technologies to improve water use efficiency and reduce water waste on the premise of maintaining the quality of agricultural products. RDI was first reported more than 40 years ago, although it has been applied in some areas, little is known about understanding of the implementation method, scope of application and detailed mechanism of RDI, resulting in the failure to achieve the effect that RDI should have. This review refers to the research on RDI in different crops published in recent years, summarizes the definition, equipment condition, function, theory illumination, plant response and application in different crops of RDI, and looks forward to its prospect. We expect that this review will provide valuable guidance for researchers and producers concerned, and support the promotion of RDI in more horticultural crops.

Metabolomics Integrated with HPLC-MS Reveals the Crucial Antioxidant Compounds of Muscadine Wine.

Wine is a kind of beverage with a variety of compounds beneficial to human health, which makes it popular all over the world and it contributes importantly to economics. The excessive oxidation of wine has always been a major problem in wine production and storage. Unlike traditional wines which are made from Eurasian grapes, wines made from muscadine grapes (Muscadinia rotundifolia Michx.) can maintain their sensory qualities under natural oxidation conditions for relatively long periods of time despite the insight mechanisms still being unclear. In this study, two muscadine wines, Carlos (CAL) and Noble (NOB), and two traditional wines, Chardonnay (CH) and Marselan (MAS), were chosen for comparison of their compositional alteration during oxidation, in order to analyze the principal components contributing to the antioxidant characteristics of muscadine wines. The DPPH, ORAC, color intensity, and total phenolic content changes during the natural oxidation process were analyzed. Six core significantly changed metabolites (SCMs, avicularin, beta-lactose, delphinidin-3-O-glucoside, ellagic acid, myricetin, and 4-methylcatechol [p < 0.05]) related to the oxidation process were determined. In addition, HPLC−MS was also used to identify pyrogallol which is a unique antioxidant compound in muscadine wine. The present work aims to reveal the crucial antioxidant compounds of muscadine wine and provide valuable information and a new platform for future research on wine oxidation.

Genomic insights into biocontrol potential of Bacillus stercoris LJBS06.

Bacillus spp. have been widely reported with the ability to control plant diseases. In this work, we analyzed the whole genome of LJBS06, which was isolated from grapevine rhizosphere soil. In view of physiological and biochemical characteristics, genome data, and phylogenetic analysis of 16S rRNA, LJBS06 was affiliated with Bacillus stercoris. LJBS06 showed antagonistic activities against a variety of plant pathogens. The inhibition rate of Magnaporthe oryzae was up to 75.05% and the inhibition rates of Colletotrichum gloeosporioides, Coniothyrium diplodiella, and Botrytis cinerea were all above 50% in the plate assays. The genome of LJBS06 had a 4,154,362-bp circular chromosome, with an average GC content of 43.96%, containing an 82,935-bp plasmid with a GC content of 35.18%. The circular chromosome of LJBS06 contained 4231 protein-coding genes, 30 rRNA genes, and 87 tRNA genes, including genes related to the synthesis of plant defense-related enzymes and the promotion of plant growth. Meanwhile, 11 gene clusters involved in biosynthesis of secondary metabolites were present in the genome of LJBS06. In conclusion, our findings indicated that LJBS06 strain had the necessary genetic machinery to control plant pathogens and provided insights for future studies of the biocontrol mechanisms of B. stercoris LJBS06. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-03000-6.

Comprehensive Leaf Cell Wall Analysis Using Carbohydrate Microarrays Reveals Polysaccharide-Level Variation between Vitis Species with Differing Resistance to Downy Mildew.

The cell wall acts as one of the first barriers of the plant against various biotic stressors. Previous studies have shown that alterations in wall polysaccharides may influence crop disease resistance. In the grapevine family, several native species (e.g., Chinese wild grapevine) show a naturally higher resistance to microbial pathogens than cultivated species (e.g., Vitis vinifera), and this trait could be inherited through breeding. Despite the importance of the cell wall in plant immunity, there are currently no comprehensive cell wall profiles of grapevine leaves displaying differing resistance phenotypes, due to the complex nature of the cell wall and the limitations of analytical techniques available. In this study, the cutting-edge comprehensive carbohydrate microarray technology was applied to profile uninfected leaves of the susceptible cultivar (Vitis vinifera cv. "Cabernet Sauvignon"), a resistant cultivar (Vitis amurensis cv. "Shuanghong") and a hybrid offspring cross displaying moderate resistance. The microarray approach uses monoclonal antibodies, which recognize polysaccharides epitopes, and found that epitope abundances of highly esterified homogalacturonan (HG), xyloglucan (with XXXG motif), (galacto)(gluco)mannan and arabinogalactan protein (AGP) appeared to be positively correlated with the high resistance of Vitis amurensis cv. "Shuanghong" to mildew. The quantification work by gas chromatography did not reveal any significant differences for the monosaccharide constituents, suggesting that polysaccharide structural alterations may contribute more crucially to the resistance observed; this is again supported by the contact infrared spectroscopy of cell wall residues, revealing chemical functional group changes (e.g., esterification of pectin). The identification of certain wall polysaccharides that showed alterations could be further correlated with resistance to mildew. Data from the use of the hybrid material in this study have preliminarily suggested that these traits could be inherited and may be applied as potential structural biomarkers in future breeding work.

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.

Plasmopara viticola effector PvRXLR111 stabilizes VvWRKY40 to promote virulence.

Plasmopara viticola, the causal organism of grapevine downy mildew, secretes a vast array of effectors to manipulate host immunity. Previously, several cell death-inducing PvRXLR effectors have been identified, but their functions and host targets are poorly understood. Here, we investigated the role of PvRXLR111, a cell death-inducing RXLR effector, in manipulating plant immunity. When coexpressed with other PvRXLR effectors, PvRXLR111-induced cell death was prevented. Transient expression of PvRXLR111 in Nicotiana benthamiana suppressed bacterial flagellin peptide flg22-elicited immune responses and enhanced Phytophthora capsici infection. PvRXLR111 induction in Arabidopsis increased susceptibility to Hyaloperonospora arabidopsidis. PvRXLR111 expression in Pseudomonas syringae promoted bacterial colonization. By immunoprecipitation-mass spectrometry analysis, yeast two-hybrid, pull-down, and bimolecular fluorescence complementation assays, it was shown that PvRXLR111 interacted with Vitis vinifera putative WRKY transcription factor 40 (VvWRKY40), which increased VvWRKY40 stability. Transient expression of VvWRKY40 in N. benthamiana inhibited flg22-induced reactive oxygen species burst and enhanced P. capsici infection and silencing NbWRKY40 attenuated P. capsici colonization. These results suggest VvWRKY40 functions as a negative regulator in plant immunity and that PvRXLR111 suppresses host immunity by stabilizing VvWRKY40.

Identifying Plasmopara viticola resistance Loci in grapevine (Vitis amurensis) via genotyping-by-sequencing-based QTL mapping.

Downy mildew, caused by Plasmopara viticola, is a major disease that affects grapevines, and a few resistance (R) genes have been identified thus far. In order to identify R genes, we investigated F1 progeny from a cross between the downy mildew-resistant Vitis amurensis 'Shuang Hong' and the susceptible Vitis vinifera 'Cabernet Sauvignon'. The P. viticola-resistance of the progeny varied continuously and was segregated as a quantitative trait. Genotyping-by-sequencing was used to construct linkage maps. The integrated map spanned 1898.09 cM and included 5603 single nucleotide polymorphisms on 19 linkage groups (LGs). Linkage analysis identified three quantitative trait loci (QTLs) for P. viticola resistance: 22 (Rpv22) on LG 02, Rpv23 on LG15, and Rpv24 on LG18. The phenotypic variance contributed by these three QTLs ranged from 15.9 to 30.0%. qRT-PCR analysis of R-gene expression in these QTLs revealed a CC-NBS-LRR disease resistance gene RPP8, two LRR receptor-like serine/threonine-protein kinases, a serine/threonine-protein kinase BLUS1, a glutathione peroxidase 8, an ethylene-responsive transcription factor ERF038, and a transcription factor bZIP11 were induced by P. viticola, and these genes may play important role in P. viticola response.

Cgr1, a ripe rot resistance QTL in Vitis amurensis 'Shuang Hong' grapevine.

Ripe rot is a serious grapevine disease in Vitis L. and Muscadinia (Planch.) Small. However, resistance to this disease has been reported in some oriental Vitis species. To identify resistance-related Quantitative Trait Loci (QTLs) from the Chinese grape species V. amurensis, an F1 population of V. vinifera 'Cabernet Sauvignon' × V. amurensis 'Shuang Hong' was used to map the ripe rot resistance loci expected in 'Shuang Hong' grape. A total of 7598 single nucleotide polymorphisms (SNPs) between the parents were identified in our previous study, and 934 SNPs were selected for genetic map construction. These SNPs are distributed across the 19 chromosomes covering a total of 1665.31 cM in length, with an average of 1.81 cM between markers. Ripe rot resistance phenotypes among the hybrids were evaluated in vitro using excised leaves for three consecutive years from 2016 to 2018; a continuous variation was found among the F1 hybrids, and the Pearson correlation coefficients of the phenotypes scored in all three years were significant at the 0.01 level. Notably, the first QTL reported for resistance to grape ripe rot disease, named Cgr1, was identified on chromosome 14 of 'Shuang Hong' grapevine. Cgr1 could explain up to 19.90% of the phenotypic variance. In addition, a SNP named 'np19345' was identified as a molecular marker closely linked to the peak of Cgr1 and has the potential to be developed as a marker for the Cgr1 resistance haplotype.

The complete chloroplast genome sequence of Vitis davidii Foex strain 'SJTU003'.

In the present study, the complete chloroplast genome of Vitis davidii Foex strain 'SJTU003' was assembled and subjected to phylogenetic analysis. This chloroplast genome of 'SJTU003' was 161,335 bp in length, including two inverted repeat regions (IRa and IRb) that were separated by a large single-copy region (89,570 bp) and a small single-copy region (19,059 bp). The genome contained 133 genes, including 88 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. Phylogenetic analysis indicated that V. davidii is most closely related to Vitis flexuosa and Vitis amurensis.

The complete chloroplast genome sequence of Vitis pseudoreticulata.

Vitis pseudoreticulata is a wild Vitis species distributed in Southern China and wildly used to crossbreeding based on its resistance to moisture. In this study, the complete chloroplast genome of V. pseudoreticulata was assembled for the first time. The chloroplast genome was 161,065 bp in length, including two single-copy regions (19,083 and 89,276), which separated by a pair of inverted repeat regions. In totally, 132 genes were predicted, including 87 CDSs, 8 rRNA genes and 37 tRNA genes. The phylogenetic tree analysis showed that V. pseudoreticulata was the closest related to Vitis amurensis.

Ectopic Expression of Grapevine Gene VaRGA1 in Arabidopsis Improves Resistance to Downy Mildew and Pseudomonas syringae pv. tomato DC3000 But Increases Susceptibility to Botrytis cinerea.

The protein family with nucleotide binding sites and leucine-rich repeat (NBS-LRR) in plants stimulates immune responses caused by effectors and can mediate resistance to hemi-biotrophs and biotrophs. In our previous study, a Toll-interleukin-1(TIR)-NBS-LRR gene cloned from Vitis amurensis "Shuanghong", VaRGA1, was induced by Plasmopara viticola and could improve the resistance of tobacco to Phytophthora capsici. In this study, VaRGA1 in "Shuanghong" was also induced by salicylic acid (SA), but inhibited by jasmonic acid (JA). To investigate whether VaRGA1 confers broad-spectrum resistance to pathogens, we transferred this gene into Arabidopsis and then treated with Hyaloperonospora arabidopsidis (Hpa), Botrytis cinerea (B. cinerea), and Pseudomonas syringae pv. tomato DC3000 (PstDC3000). Results showed that VaRGA1 improved transgenic Arabidopsis thaliana resistance to the biotrophic Hpa and hemi-biotrophic PstDC3000, but decreased resistance to the necrotrophic B. cinerea. Additionally, qPCR assays showed that VaRGA1 plays an important role in disease resistance by activating SA and inhibiting JA signaling pathways. A 1104 bp promoter fragment of VaRGA1 was cloned and analyzed to further elucidate the mechanism of induction of the gene at the transcriptional level. These results preliminarily confirmed the disease resistance function and signal regulation pathway of VaRGA1, and contributed to the identification of R-genes with broad-spectrum resistance function.

Molecular Characterization and Overexpression of VpRPW8s from Vitis pseudoreticulata Enhances Resistance to Phytophthora capsici in Nicotiana benthamiana.

RPW8 genes are atypical broad-spectrum genes that provide resistance to powdery mildew, downy mildew, the cauliflower mosaic virus in Arabidopsis thaliana, and powdery mildew in tobacco. They play important roles in basal plant pathogen defense. They also provide insights into a novel disease resistance mechanism. In this study, we report on homologous RPW8 genes in Vitis pseudoreticulata. Five VpRPW8 genes were cloned; their Open Reading Frame (ORF) sequences ranged from 1994 base pairs to 2478 base pairs. They were comprised of five exons and four introns and shared 78.66% identity. Their proteins had typical conserved RPW8 and NB-LRR (the nucleotide-binding site and the leucine-rich repeats) domains (except VpRPW8-d, which lacked LRR domains). Prokaryotic expression results were consistent with predicted molecular weights. All five RPW8 genes were located in the cytoplasm. Quantitative real-time PCR (qRT-PCR) analysis showed that VpRPW8s in V. pseudoreticulata were induced by Plasmopara viticola, but nearly only VvRPW8-d genes were induced in Vitis vinifera. Furthermore, a VpRPW8 transgenic tobacco system was established. Overexpressed VpRPW8s enhanced resistance to Phytophthora capsici and VpRPW8s conferred varying degrees of resistance to Ph. capsici in Nicotiana benthamiana. Our study presents novel members of the plant RPW8 family and suggests that VpRPW8s are involved in enhanced resistance to P. viticola and Ph. capsici.