Chimeric mutations in grapevine ENHANCED DISEASE RESISTANCE1 improve resistance to powdery mildew without growth penalty.

Grapevine (Vitis vinifera L.) incurs severe quality degradation and yield loss from powdery mildew, a major fungal disease caused by Erysiphe necator. ENHANCED DISEASE RESISTANCE1 (EDR1), a Raf-like mitogen-activated protein kinase kinase kinase, negatively regulates defense responses against powdery mildew in Arabidopsis (Arabidopsis thaliana). However, little is known about the role of the putatively orthologous EDR1 gene in grapevine. In this study, we obtained grapevine VviEDR1-edited lines using CRISPR/Cas9. Plantlets containing homozygous and bi-allelic indels in VviEDR1 developed leaf lesions shortly after transplanting into the soil and died at the seedling stage. Transgenic plants expressing wild-type VviEDR1 and mutant Vviedr1 alleles as chimera (designated as VviEDR1-chi) developed normally and displayed enhanced resistance to powdery mildew. Interestingly, VviEDR1-chi plants maintained a spatiotemporally distinctive pattern of VviEDR1 mutagenesis: while almost no mutations were detected from terminal buds, ensuring normal function of the apical meristem, mutations occurred in young leaves and increased as leaves matured, resulting in resistance to powdery mildew. Further analysis showed that the resistance observed in VviEDR1-chi plants was associated with callose deposition, increased production of salicylic acid and ethylene, H2O2 production and accumulation, and host cell death. Surprisingly, no growth penalty was observed with VviEDR1-chi plants. Hence, this study demonstrated a role of VviEDR1 in the negative regulation of resistance to powdery mildew in grapevine and provided an avenue for engineering powdery mildew resistance in grapevine.

Transcription factors VviWRKY10 and VviWRKY30 co-regulate powdery mildew resistance in grapevine.

Grapevine (Vitis vinifera) is an economically important fruit crop worldwide. The widely cultivated grapevine is susceptible to powdery mildew caused by Erysiphe necator. In this study, we used CRISPR-Cas9 to simultaneously knock out VviWRKY10 and VviWRKY30 encoding two transcription factors reported to be implicated in defense regulation. We generated 53 wrky10 single mutant transgenic plants and 15 wrky10 wrky30 double mutant transgenic plants. In a 2-yr field evaluation of powdery mildew resistance, the wrky10 mutants showed strong resistance, while the wrky10 wrky30 double mutants showed moderate resistance. Further analyses revealed that salicylic acid (SA) and reactive oxygen species contents in the leaves of wrky10 and wrky10 wrky30 were substantially increased, as was the ethylene (ET) content in the leaves of wrky10. The results from dual luciferase reporter assays, electrophoretic mobility shift assays and chromatin immunoprecipitation (ChIP) assays demonstrated that VviWRKY10 could directly bind to the W-boxes in the promoter of SA-related defense genes and inhibit their transcription, supporting its role as a negative regulator of SA-dependent defense. By contrast, VviWRKY30 could directly bind to the W-boxes in the promoter of ET-related defense genes and promote their transcription, playing a positive role in ET production and ET-dependent defense. Moreover, VviWRKY10 and VviWRKY30 can bind to each other's promoters and mutually inhibit each other's transcription. Taken together, our results reveal a complex mechanism of regulation by VviWRKY10 and VviWRKY30 for activation of measured and balanced defense responses against powdery mildew in grapevine.

Pan-Genomic Analysis Identifies the Chinese Strain as a New Subspecies of Xanthomonas fragariae.

Xanthomonas fragariae is classified as a quarantine pathogen by the European and Mediterranean Plant Protection Organization. It commonly induces typical angular leaf spot (ALS) symptoms in strawberry leaves. X. fragariae strains from China (YL19, SHAQP01, and YLX21) exhibit ALS symptoms in leaves and more severe symptoms of dry cavity rot in strawberry crowns. Conversely, strains from other countries do not cause severe dry cavity rot symptoms in strawberries. After employing multilocus sequence analysis (MLSA), average nucleotide identity (ANI), and amino acid identity (AAI), we determined that Chinese strains of X. fragariae are genetically distinct from other strains and can be considered a new subspecies. Subsequent analysis of 63 X. fragariae genomes published at NCBI using IPGA and EDGAR3.0 revealed the pan-genomic profile, with 1,680 shared genes present in all 63 strains, including 71 virulence-related genes. Additionally, we identified 123 genes exclusive to all the Chinese strains, encompassing 12 virulence-related genes. The qRT-PCR analysis demonstrated that the expression of XopD, XopG1, CE8, GT2, and GH121 out of 12 virulence-related genes of Chinese strains (YL19) exhibited a constant increase in the early stages (6, 24, 54, and 96 hours postinoculation [hpi]) of strawberry leaf infected by YL19. So, the presence of XopD, XopG1, CE8, GT2, and GH121 in Chinese strains may play important roles in the early infection process of Chinese strains. These findings offer novel insights into comprehending the population structure and variation in the pathogenic capacity of X. fragariae.

An effector of Erysiphe necator translocates to chloroplasts and plasma membrane to suppress host immunity in grapevine.

The powdery mildew (Erysiphe necator) is a prevalent pathogen hampering grapevine growth in the vineyard. An arsenal of candidate secreted effector proteins (CSEPs) was encoded in the E. necator genome, but it is largely unclear what role CSEPs plays during the E. necator infection. In the present study, we identified a secreted effector CSEP080 of E. necator, which was located in plant chloroplasts and plasma membrane. Transient expressing CSEP080 promotes plant photosynthesis and inhibits INF1-induced cell death in tobacco leaves. We found that CSEP080 was a necessary effector for the E. necator pathogenicity, which interacted with grapevine chloroplast protein VviB6f (cytochrome b6-f complex iron-sulfur subunit), affecting plant photosynthesis. Transient silencing VviB6f increased the plant hydrogen peroxide production, and the plant resistance to powdery mildew. In addition, CSEP080 manipulated the VviPE (pectinesterase) to promote pectin degradation. Our results demonstrated the molecular mechanisms that an effector of E. necator translocates to host chloroplasts and plasma membrane, which suppresses with the grapevine immunity system by targeting the chloroplast protein VviB6f to suppress hydrogen peroxide accumulation and manipulating VviPE to promote pectin degradation.

Systemic Colonization of Xanthomonas fragariae Strain YL19 Causing Dry Cavity Rot of Strawberry Crown Tissue in China.

Xanthomonas fragariae usually causes angular leaf spot (ALS) of strawberry, a serious bacterial disease in many strawberry-producing regions worldwide. Recently, a new strain of X. fragariae (YL19) was isolated from strawberry in China and has been shown to cause dry cavity rot in strawberry crown. In this study, we constructed a green fluorescent protein (GFP)-labeled Xf YL19 (YL19-GFP) to visualize the infection process and pathogen colonization in strawberries. Foliar inoculation of YL19-GFP resulted in the pathogen migrating from the leaves to the crown, whereas dip inoculation of wounded crowns or roots resulted in the migration of bacteria from the crowns or roots to the leaves. These two invasion types both resulted in the systematic spread of YL19-GFP, but inoculation of a wounded crown was more harmful to the strawberry plant than foliar inoculation. Results increased our understanding of the systemic invasion of X. fragariae, and the resultant crown cavity caused by Xf YL19.

A point mutation in the gene encoding magnesium chelatase I subunit influences strawberry leaf color and metabolism.

Magnesium chelatase (MgCh) catalyzes the insertion of magnesium into protoporphyrin IX, a vital step in chlorophyll (Chl) biogenesis. The enzyme consists of 3 subunits, MgCh I subunit (CHLI), MgCh D subunit (CHLD), and MgCh H subunit (CHLH). The CHLI subunit is an ATPase that mediates catalysis. Previous studies on CHLI have mainly focused on model plant species, and its functions in other species have not been well described, especially with regard to leaf coloration and metabolism. In this study, we identified and characterized a CHLI mutant in strawberry species Fragaria pentaphylla. The mutant, noted as p240, exhibits yellow-green leaves and a low Chl level. RNA-Seq identified a mutation in the 186th amino acid of the CHLI subunit, a base conserved in most photosynthetic organisms. Transient transformation of wild-type CHLI into p240 leaves complemented the mutant phenotype. Further mutants generated from RNA-interference (RNAi) and CRISPR/Cas9 gene editing recapitulated the mutant phenotype. Notably, heterozygous chli mutants accumulated more Chl under low light conditions compared with high light conditions. Metabolite analysis of null mutants under high light conditions revealed substantial changes in both nitrogen and carbon metabolism. Further analysis indicated that mutation in Glu186 of CHLI does not affect its subcellular localization nor the interaction between CHLI and CHLD. However, intramolecular interactions were impaired, leading to reduced ATPase and MgCh activity. These findings demonstrate that Glu186 plays a key role in enzyme function, affecting leaf coloration via the formation of the hexameric ring itself, and that manipulation of CHLI may be a means to improve strawberry plant fitness and photosynthetic efficiency under low light conditions.

Genome Sequence Resource for Erysiphe necator NAFU1, a Grapevine Powdery Mildew Isolate Identified in Shaanxi Province of China.

Erysiphe necator is an economically important biotrophic fungal pathogen responsible for powdery mildew disease on grapevine. Currently, genome sequences are available for only a few E. necator isolates from the United States. Based on the combination of Nanopore and Illumina sequencing technologies, we present here the complete genome assembly for an isolate of E. necator, NAFU1, identified in China. We acquired a total of 15.93 Gb of raw reads. These reads were processed into a 61.12-Mb genome assembly containing 73 contigs with an N50 of 2.06 Mb and a maximum length of 6.05 Mb. Combining the results of three gene-prediction modules (i.e., an evidence-based gene modeler [EVidenceModeler], an ab initio gene modeler, and a homology-based gene modeler), we predicted 7,235 protein-coding genes in the assembled genome of E. necator NAFU1. This information will facilitate studies of genome evolution and pathogenicity mechanisms of E. necator and other powdery mildew species through comparative genome sequence analysis and other molecular genetic tools.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Overexpression of two CDPKs from wild Chinese grapevine enhances powdery mildew resistance in Vitis vinifera and Arabidopsis.

Calcium-dependent protein kinases (CDPKs) play vital roles in metabolic regulations and stimuli responses in plants. However, little is known about their function in grapevine. Here, we report that VpCDPK9 and VpCDPK13, two paralogous CDPKs from Vitis pseudoreticulata accession Baihe-35-1, appear to positively regulate powdery mildew resistance. The transcription of them in leaves of 'Baihe-35-1' were differentially induced upon powdery mildew infection. Overexpression of VpCDPK9-YFP or VpCDPK13-YFP in the V. vinifera susceptible cultivar Thompson Seedless resulted in enhanced resistance to powdery mildew (YFP, yellow fluorescent protein). This might be due to elevation of SA and ethylene production, and excess accumulation of H2 O2 and callose in penetrated epidermal cells and/or the mesophyll cells underneath. Ectopic expression of VpCDPK9-YFP in Arabidopsis resulted in varied degrees of reduced stature, pre-mature senescence and enhanced powdery mildew resistance. However, these phenotypes were abolished in VpCDPK9-YFP transgenic lines impaired in SA signaling (pad4sid2) or ethylene signaling (ein2). Moreover, both of VpCDPK9 and VpCDPK13 were found to interact with and potentially phosphorylate VpMAPK3, VpMAPK6, VpACS1 and VpACS2 in vivo (ACS, 1-aminocyclopropane-1-carboxylic acid (ACC) synthase; MAPK, mitogen-activated protein kinase). These results suggest that VpCDPK9 and VpCDPK13 contribute to powdery mildew resistance via positively regulating SA and ethylene signaling in grapevine.

CRISPR/Cas9-mediated mutagenesis of VvMLO3 results in enhanced resistance to powdery mildew in grapevine (Vitis vinifera).

Grapevine (Vitis vinifera), one of the most economically important fruit crops in the world, suffers significant yield losses from powdery mildew, a major fungal disease caused by Erysiphe necator. In addition to suppressing host immunity, phytopathogens modulate host proteins termed susceptibility (S) factors to promote their proliferation in plants. In this study, CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) technology was used to enable the targeted mutagenesis of MLO (mildew resistance Locus O) family genes that are thought to serve as S factors for powdery mildew fungi. Small deletions or insertions were induced in one or both alleles of two grapevine MLO genes, VvMLO3 and VvMLO4, in the transgenic plantlets of the powdery mildew-susceptible cultivar Thompson Seedless. The editing efficiency achieved with different CRISPR/Cas9 constructs varied from 0 to 38.5%. Among the 20 VvMLO3/4-edited lines obtained, one was homozygous for a single mutation, three harbored biallelic mutations, seven were heterozygous for the mutations, and nine were chimeric, as indicated by the presence of more than two mutated alleles in each line. Six of the 20 VvMLO3/4-edited grapevine lines showed normal growth, while the remaining lines exhibited senescence-like chlorosis and necrosis. Importantly, four VvMLO3-edited lines showed enhanced resistance to powdery mildew, which was associated with host cell death, cell wall apposition (CWA) and H2O2 accumulation. Taken together, our results demonstrate that CRISPR/Cas9 genome-editing technology can be successfully used to induce targeted mutations in genes of interest to improve traits of economic importance, such as disease resistance in grapevines.

Transcription factor FvTCP9 promotes strawberry fruit ripening by regulating the biosynthesis of abscisic acid and anthocyanins.

The plant-specific transcription factor TEOSINTE BRANCHED 1, CYCLOIDEA, and PROLIFERATING4 CELL FACTORS (TCP) plays a crucial role in plant growth and development. However, there have been no studies reporting on the function of strawberry TCP in regulating fruit development. In this study, FvTCP9, a woodland strawberry (Fragaria vesca) TCP gene, was isolated to explore its function in fruit ripening. The transcript accumulation levels of FvTCP9 were high in fruits, specifically in red fruits compared with other tissues or organs. Transient expression of the FvTCP9 gene in cultivated strawberry fruits revealed that over-expression of FvTCP9 promoted fruit ripening. Meanwhile, silencing FvTCP9, using tobacco rattle virus-induced gene silencing (VIGS), inhibited fruit ripening. The changes in ripening-related physiological conditions in transient fruits, such as the accumulation of anthocyanins and abscisic acid (ABA), and fruit firmness confirmed above results. Results suggested that FvTCP9 was involved in the biosynthesis of ABA and anthocyanins to regulate fruit ripening. Transcription analysis showed that the expression levels of ABA signaling-related genes (FaNCED1, FaPYR1, FaSnRK2, and FaABI5) were affected by FvTCP9. A yeast two-hybrid assay revealed that FvTCP9 interacted physically with FaMYC1 to modulate the biosynthesis process of anthocyanins. Taken together, this study demonstrated that FvTCP9 promoted fruit ripening by regulating the biosynthesis of ABA and anthocyanins.

The cytological basis of powdery mildew resistance in wild Chinese Vitis species.

The wild Chinese grapevines (Vitis spp.) show varying levels of resistance to powdery mildew caused by Erysiphe necator that is an economically important disease of cultivated grapevines (Vitis vinifera). However, little information is available regarding the cytological mechanisms of powdery mildew resistance in these wild relatives. Here, we studied the cytological responses of three wild Chinese grapevine accessions after they were infected with E. necator (En) NAFU1 in comparison to the susceptible V. vinifera cv. 'Thompson Seedless' grape. The hyphal growth and sporulation of En NAFU1 were significantly restricted in wild species compared to 'Thompson Seedless', which appears to be associated with early cell wall deposition at the attempt sites, encasement of haustoria, and hypersensitive response-like cell death of penetrated epidermal cells. Moreover, endogenous free salicylic acid (SA) was more abundant in wild Chinese Vitis species than in 'Thompson Seedless' under pathogen-free condition. During En NAFU1 colonization, SA conjugates accumulated higher in wild grapevines than in 'Thompson Seedless'. In addition, the species-specific expression patterns of defense-associated genes during En NAFU1 colonization indicated that mechanisms underlying powdery mildew resistance are divergent among different wild Chinese Vitis species. These results contribute to understanding of mechanisms underlying defense responses of wild Chinese Vitis species against powdery mildew.

Ectopic expression of Arabidopsis broad-spectrum resistance gene RPW8.2 improves the resistance to powdery mildew in grapevine (Vitis vinifera).

Powdery mildew is the most economically important disease of cultivated grapevines worldwide. Here, we report that the Arabidopsis broad-spectrum disease resistance gene RPW8.2 could improve resistance to powdery mildew in Vitis vinifera cv. Thompson Seedless. The RPW8.2-YFP fusion gene was stably expressed in grapevines from either the constitutive 35S promoter or the native promoter (NP) of RPW8.2. The grapevine shoots and plantlets transgenic for 35S::RPW8.2-YFP showed reduced rooting and reduced growth at later development stages in the absence of any pathogens. Infection tests with an adapted grapevine powdery mildew isolate En NAFU1 showed that hyphal growth and sporulation were significantly restricted in transgenic grapevines expressing either of the two constructs. The resistance appeared to be attributable to the ectopic expression of RPW8.2, and associated with the enhanced encasement of the haustorial complex (EHC) and onsite accumulation of H2O2. In addition, the RPW8.2-YFP fusion protein showed focal accumulation around the fungal penetration sites. Transcriptome analysis revealed that ectopic expression of RPW8.2 in grapevines not only significantly enhanced salicylic acid-dependent defense signaling, but also altered expression of other phytohormone-associated genes. Taken together, our results indicate that RPW8.2 could be utilized as a transgene for improving resistance against powdery mildew in grapevines.

Identification and expression analysis of heat shock transcription factors in the wild Chinese grapevine (Vitis pseudoreticulata).

Heat shock transcription factors (Hsfs) are known to play pivotal roles in the adaptation of plants to heat stress and other stress stimuli. While grapevine (Vitis vinifera L.) is one of the most important fruit crops worldwide, little is known about the Hsf family in Vitis spp. Here, we identified nineteen putative Hsf genes (VviHsfs) in Vitis spp based on the 12 × grape genome (V. vinifera L.). Phylogenetic analysis revealed three classes of grape Hsf genes (classes A, B, and C). Additional comparisons between grape and Arabidopsis thaliana demonstrated that several VviHsfs genes occurred in corresponding syntenic blocks of Arabidopsis. Moreover, we examined the expression profiles of the homologs of the VviHsfs genes (VpHsfs) in the wild Chinese Vitis pseudoreticulata accession Baihe-35-1, which is tolerant to various environmental stresses. Among the nineteen VpHsfs, ten VpHsfs displayed lower transcript levels under non-stress conditions and marked up-regulation during heat stress treatment; several VpHsfs also displayed altered expression levels in response to cold, salt, and hormone treatments, suggesting their versatile roles in response to stress stimuli. In addition, eight VpHsf-GFP fusion proteins showed differential subcellular localization in V. pseudoreticulata mesophyll protoplasts. Taken together, our data may provide an important reference for further studies of Hsf genes in Vitis spp.

Identification and utilization of a new Erysiphe necator isolate NAFU1 to quickly evaluate powdery mildew resistance in wild Chinese grapevine species using detached leaves.

The most economically important disease of cultivated grapevines worldwide is powdery mildew caused by the biotrophic fungal pathogen Erysiphe necator. To integrate effective genetic resistance into cultivated grapevines, numerous disease resistance screens of diverse Vitis germplasm, including wild species, have been conducted to identify powdery mildew resistance, but the results have been inconsistent. Here, a new powdery mildew isolate that is infectious on grapevines, designated Erysiphe necator NAFU1 (En. NAFU1), was identified and characterized by phylogeny inferred from the internal transcribed spacer (ITS) of pathogen ribosomal DNA sequences. Three classical methods were compared for the maintenance of En. NAFU1, and the most convenient method was maintenance on detached leaves and propagation by contact with infected leaves. Furthermore, controlled inoculations of En. NAFU1 were performed using detached leaves from 57 wild Chinese grapevine accessions to quickly evaluate powdery mildew resistance based on trypan blue staining of leaf sections. The results were compared with previous natural epidemics in the field. Among the screened accessions inoculated with En. NAFU1, 22.8% were resistant, 33.3% were moderately resistant, and 43.9% were susceptible. None of the accessions assessed herein were immune from infection. These results support previous findings documenting the presence of race-specific resistance to E. necator in wild Chinese grapevine. The resistance of wild Chinese grapevine to En. NAFU1 could be due to programmed cell death. The present results suggest that En. NAFU1 isolate could be used for future large-scale screens of resistance to powdery mildew in diverse Vitis germplasms and investigations of the interaction between grapevines and pathogens.

Genome-wide Identification and Expression Analysis of the CDPK Gene Family in Grape, Vitis spp.

BACKGROUND: Calcium-dependent protein kinases (CDPKs) play vital roles in plant growth and development, biotic and abiotic stress responses, and hormone signaling. Little is known about the CDPK gene family in grapevine. RESULTS: In this study, we performed a genome-wide analysis of the 12X grape genome (Vitis vinifera) and identified nineteen CDPK genes. Comparison of the structures of grape CDPK genes allowed us to examine their functional conservation and differentiation. Segmentally duplicated grape CDPK genes showed high structural conservation and contributed to gene family expansion. Additional comparisons between grape and Arabidopsis thaliana demonstrated that several grape CDPK genes occured in the corresponding syntenic blocks of Arabidopsis, suggesting that these genes arose before the divergence of grapevine and Arabidopsis. Phylogenetic analysis divided the grape CDPK genes into four groups. Furthermore, we examined the expression of the corresponding nineteen homologous CDPK genes in the Chinese wild grape (Vitis pseudoreticulata) under various conditions, including biotic stress, abiotic stress, and hormone treatments. The expression profiles derived from reverse transcription and quantitative PCR suggested that a large number of VpCDPKs responded to various stimuli on the transcriptional level, indicating their versatile roles in the responses to biotic and abiotic stresses. Moreover, we examined the subcellular localization of VpCDPKs by transiently expressing six VpCDPK-GFP fusion proteins in Arabidopsis mesophyll protoplasts; this revealed high variability consistent with potential functional differences. CONCLUSIONS: Taken as a whole, our data provide significant insights into the evolution and function of grape CDPKs and a framework for future investigation of grape CDPK genes.