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

Grapevine (Vitis vinifera L.) incurs severequality 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 (MAPKKK), 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 (SA) and ethylene (ET), 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.

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.

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.

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.

Ectopic expression of FvWRKY42, a WRKY transcription factor from the diploid woodland strawberry (Fragaria vesca), enhances resistance to powdery mildew, improves osmotic stress resistance, and increases abscisic acid sensitivity in Arabidopsis.

WRKY transcription factors play a critical role in biotic and abiotic stress responses in plants, but very few WRKYs have been reported in strawberry plants. Here, a multiple stress-inducible gene, FvWRKY42, was isolated from the wild diploid woodland strawberry (accession Heilongjiang-3). FvWRKY42 expression was induced by treatment with powdery mildew, salt, drought, salicylic acid (SA), methyl jasmonate (MeJA), abscisic acid (ABA), and ethylene. The protein interaction network analysis showed that the FvWRKY42 protein interacts with various stress-related proteins. Overexpression of FvWRKY42 in Arabidopsis resulted in cell death, sporulation, slow hypha growth, and enhanced resistance to powdery mildew that was concomitant with increased expression of PR1 genes in Arabidopsis. Overexpression also led to enhanced salt and drought stress tolerance, increased primary root length and germination rate, decreased water loss rate, reduced relative electrolyte leakage, and malondialdehyde accumulation, and upregulation of superoxide dismutase and catalase activity. Additionally, FvWRKY42-overexpressing Arabidopsis plants showed increased ABA sensitivity during seed germination and seedling growth, increased stomatal closure after ABA and drought treatment, and altered expression of ABA-responsive genes. Collectively, our data demonstrate that FvWRKY42 may play an important role in powdery mildew infection and the regulation of salt and drought stress responses in plants.

The WRKY transcription factors in the diploid woodland strawberry Fragaria vesca: Identification and expression analysis under biotic and abiotic stresses.

WRKY proteins comprise a large family of transcription factors that play important roles in response to biotic and abiotic stresses and in plant growth and development. To date, little is known about the WRKY gene family in strawberry. In this study, we identified 62 WRKY genes (FvWRKYs) in the wild diploid woodland strawberry (Fragaria vesca, 2n = 2x = 14) accession Heilongjiang-3. According to the phylogenetic analysis and structural features, these identified strawberry FvWRKY genes were classified into three main groups. In addition, eight FvWRKY-GFP fusion proteins showed distinct subcellular localizations in Arabidopsis mesophyll protoplasts. Furthermore, we examined the expression of the 62 FvWRKY genes in 'Heilongjiang-3' under various conditions, including biotic stress (Podosphaera aphanis), abiotic stresses (drought, salt, cold, and heat), and hormone treatments (abscisic acid, ethephon, methyl jasmonate, and salicylic acid). The expression levels of 33 FvWRKY genes were upregulated, while 12 FvWRKY genes were downregulated during powdery mildew infection. FvWRKY genes responded to drought and salt treatment to a greater extent than to temperature stress. Expression profiles derived from quantitative real-time PCR suggested that 11 FvWRKY genes responded dramatically to various stimuli at the transcriptional level, indicating versatile roles in responses to biotic and abiotic stresses. Interaction networks revealed that the crucial pathways controlled by WRKY proteins may be involved in the differential response to biotic stress. Taken together, the present work may provide the basis for future studies of the genetic modification of WRKY genes for pathogen resistance and stress tolerance in strawberry.

Identification and Transcript Analysis of the TCP Transcription Factors in the Diploid Woodland Strawberry Fragaria vesca.

Plant-specific TEOSINTE BRANCHED 1, CYCLOIDEA, and PROLIFERATING CELL FACTORS (TCP) transcription factors play versatile functions in multiple processes of plant growth and development. However, no systematic study has been performed in strawberry. In this study, 19 FvTCP genes were identified in the diploid woodland strawberry (Fragaria vesca) accession Heilongjiang-3. Phylogenetic analysis suggested that the FvTCP genes were classified into two main classes, with the second class further divided into two subclasses, which was supported by the exon-intron organizations and the conserved motif structures. Promoter analysis revealed various cis-acting elements related to growth and development, hormone and/or stress responses. We analyzed FvTCP gene transcript accumulation patterns in different tissues and fruit developmental stages. Among them, 12 FvTCP genes exhibited distinct tissue-specific transcript accumulation patterns. Eleven FvTCP genes were down-regulated in different fruit developmental stages, while five FvTCP genes were up-regulated. Transcripts of FvTCP genes also varied with different subcultural propagation periods and were induced by hormone treatments and biotic and abiotic stresses. Subcellular localization analysis showed that six FvTCP-GFP fusion proteins showed distinct localizations in Arabidopsis mesophyll protoplasts. Notably, transient over-expression of FvTCP9 in strawberry fruits dramatically affected the expression of a series of genes implicated in fruit development and ripening. Taken together, the present study may provide the basis for functional studies to reveal the role of this gene family in strawberry growth and development.

Identification, isolation, and expression analysis of heat shock transcription factors in the diploid woodland strawberry Fragaria vesca.

Heat shock transcription factors (Hsfs) are known to play dominant roles in plant responses to heat, as well as other abiotic or biotic stress stimuli. While the strawberry is an economically important fruit plant, little is known about the Hsf family in the strawberry. To explore the functions of strawberry Hsfs in abiotic and biotic stress responses, this study identified 17 Hsf genes (FvHsfs) in a wild diploid woodland strawberry (Fragaria vesca, 2n = 2x = 14) and isolated 14 of these genes. Phylogenetic analysis divided the strawberry FvHsfs genes into three main groups. The evolutionary and structural analyses revealed that the FvHsf family is conserved. The promoter sequences of the FvHsf genes contain upstream regulatory elements corresponding to different stress stimuli. In addition, 14 FvHsf-GFP fusion proteins showed differential subcellular localization in Arabidopsis mesophyll protoplasts. Furthermore, we examined the expression of the 17 FvHsf genes in wild diploid woodland strawberries under various conditions, including abiotic stresses (heat, cold, drought, and salt), biotic stress (powdery mildew infection), and hormone treatments (abscisic acid, ethephon, methyl jasmonate, and salicylic acid). Fifteen of the seventeen FvHsf genes exhibited distinct changes on the transcriptional level during heat treatment. Of these 15 FvHsfs, 8 FvHsfs also exhibited distinct responses to other stimuli on the transcriptional level, indicating versatile roles in the response to abiotic and biotic stresses. Taken together, the present work may provide the basis for further studies to dissect FvHsf function in response to stress stimuli.