SR45a plays a key role in enhancing cotton resistance to Verticillium dahliae by alternative splicing of immunity genes.

Alternative splicing (AS) of pre-mRNAs increases the diversity of transcriptome and proteome and plays fundamental roles in plant development and stress responses. However, the prevalent changes in AS events and the regulating mechanisms of plants in response to pathogens remain largely unknown. Here, we show that AS changes are an important mechanism conferring cotton immunity to Verticillium dahliae (Vd). GauSR45a, encoding a serine/arginine-rich RNA binding protein, was upregulated expression and underwent AS in response to Vd infection in Gossypium australe, a wild diploid cotton species highly resistant to Vd. Silencing GauSR45a substantially reduced the splicing ratio of Vd-induced immune-associated genes, including GauBAK1 (BRI1-associated kinase 1) and GauCERK1 (chitin elicitor receptor kinase 1). GauSR45a binds to the GAAGA motif that is commonly found in the pre-mRNA of genes essential for PTI, ETI, and defense. The binding between GauSR45a and the GAAGA motif in the pre-mRNA of BAK1 was enhanced by two splicing factors of GauU2AF35B and GauU1-70 K, thereby facilitating exon splicing; silencing either AtU2AF35B or AtU1-70 K decreased the resistance to Vd in transgenic GauSR45a Arabidopsis. Overexpressing the short splicing variant of BAK1GauBAK1.1 resulted in enhanced Verticillium wilt resistance rather than the long one GauBAK1.2. Vd-induced far more AS events were in G. barbadense (resistant tetraploid cotton) than those in G. hirsutum (susceptible tetraploid cotton) during Vd infection, indicating resistance divergence in immune responses at a genome-wide scale. We provided evidence showing a fundamental mechanism by which GauSR45a enhances cotton resistance to Vd through global regulation of AS of immunity genes.

Promotion of apoplastic oxidative burst by artificially selected GhCBSX3A enhances Verticillium dahliae resistance in upland cotton.

Verticillium wilt (VW) is a devasting disease affecting various plants, including upland cotton, a crucial fiber crop. Despite its impact, the genetic basis underlying cotton's susceptibility or defense against VW remains unclear. Here, we conducted a genome-wide association study on VW phenotyping in upland cotton and identified a locus on A13 that is significantly associated with VW resistance. We then identified a cystathionine ß-synthase domain gene at A13 locus, GhCBSX3A, which was induced by Verticillium dahliae. Functional analysis, including expression silencing in cotton and overexpression in Arabidopsis thaliana, confirmed that GhCBSX3A is a causal gene at the A13 locus, enhancing SAR-RBOHs-mediated apoplastic oxidative burst. We found allelic variation on the TATA-box of GhCBSX3A promoter attenuated its expression in upland cotton, thereby weakening VW resistance. Interestingly, we discovered that altered artificial selection of GhCBSX3A_R (an elite allele for VW) under different VW pressures during domestication and other improved processes allows specific human needs to be met. Our findings underscore the importance of GhCBSX3A in response to VW, and we propose a model for defense-associated genes being selected depending on the pathogen's pressure. The identified locus and gene serve as promising targets for VW resistance enhancement in cotton through genetic engineering.

Suppression of plant immunity by Verticillium dahliae effector Vd6317 through AtNAC53 association.

Verticillium dahliae, a soil-borne fungal pathogen, compromises host innate immunity by secreting a plethora of effectors, thereby facilitating host colonization and causing substantial yield and quality losses. The mechanisms underlying the modulation of cotton immunity by V. dahliae effectors are predominantly unexplored. In this study, we identified that the V. dahliae effector Vd6317 inhibits plant cell death triggered by Vd424Y and enhances PVX viral infection in Nicotiana benthamiana. Attenuation of Vd6317 significantly decreased the virulence of V. dahliae, whereas ectopic expression of Vd6317 in Arabidopsis and cotton enhanced susceptibility to V. dahliae infection, underscoring Vd6317's critical role in pathogenicity. We observed that Vd6317 targeted the Arabidopsis immune regulator AtNAC53, thereby impeding its transcriptional activity on the defense-associated gene AtUGT74E2. Arabidopsis nac53 and ugt74e2 mutants exhibited heightened sensitivity to V. dahliae compared to wild-type plants. A mutation at the conserved residue 193L of Vd6317 abrogated its interaction with AtNAC53 and reduced the virulence of V. dahliae, which was partially attributable to a reduction in Vd6317 protein stability. Our findings unveil a hitherto unrecognized regulatory mechanism by which the V. dahliae effector Vd6317 directly inhibits the plant transcription factor AtNAC53 activity to suppress the expression of AtUGT74E2 and plant defense.

Epigenetic regulation of nuclear processes in fungal plant pathogens.

Through the association of protein complexes to DNA, the eukaryotic nuclear genome is broadly organized into open euchromatin that is accessible for enzymes acting on DNA and condensed heterochromatin that is inaccessible. Chemical and physical alterations to chromatin may impact its organization and functionality and are therefore important regulators of nuclear processes. Studies in various fungal plant pathogens have uncovered an association between chromatin organization and expression of in planta-induced genes that are important for pathogenicity. This review discusses chromatin-based regulation mechanisms as determined in the fungal plant pathogen Verticillium dahliae and relates the importance of epigenetic transcriptional regulation and other nuclear processes more broadly in fungal plant pathogens.

Verticillium dahliae Vta3 promotes ELV1 virulence factor gene expression in xylem sap, but tames Mtf1-mediated late stages of fungus-plant interactions and microsclerotia formation.

Verticillium transcription activator of adhesion 3 (Vta3) is required for plant root colonization and pathogenicity of the soil-borne vascular fungus Verticillium dahliae. RNA sequencing identified Vta3-dependent genetic networks required for growth in tomato xylem sap. Vta3 affects the expression of more than 1,000 transcripts, including candidates with predicted functions in virulence and morphogenesis such as Egh16-like virulence factor 1 (Elv1) and Master transcription factor 1 (Mtf1). The genes encoding Elv1 and Mtf1 were deleted and their functions in V. dahliae growth and virulence on tomato (Solanum lycopersicum) plants were investigated using genetics, plant infection experiments, gene expression studies and phytohormone analyses. Vta3 contributes to virulence by promoting ELV1 expression, which is dispensable for vegetative growth and conidiation. Vta3 decreases disease symptoms mediated by Mtf1 in advanced stages of tomato plant colonization, while Mtf1 induces the expression of fungal effector genes and tomato pathogenesis-related protein genes. The levels of pipecolic and salicylic acids functioning in tomato defense signaling against (hemi-) biotrophic pathogens depend on the presence of MTF1, which promotes the formation of resting structures at the end of the infection cycle. In summary, the presence of VTA3 alters gene expression of virulence factors and tames the Mtf1 genetic subnetwork for late stages of plant disease progression and subsequent survival of the fungus in the soil.

A fungal effector suppresses the nuclear export of AGO1-miRNA complex to promote infection in plants.

Communication between interacting organisms via bioactive molecules is widespread in nature and plays key roles in diverse biological processes. Small RNAs (sRNAs) can travel between host plants and filamentous pathogens to trigger transkingdom RNA interference (RNAi) in recipient cells and modulate plant defense and pathogen virulence. However, how fungal pathogens counteract transkingdom antifungal RNAi has rarely been reported. Here we show that a secretory protein VdSSR1 (secretory silencing repressor 1) from Verticillium dahliae, a soil-borne phytopathogenic fungus that causes wilt diseases in a wide range of plant hosts, is required for fungal virulence in plants. VdSSR1 can translocate to plant nucleus and serve as a general suppressor of sRNA nucleocytoplasmic shuttling. We further reveal that VdSSR1 sequesters ALY family proteins, adaptors of the TREX complex, to interfere with nuclear export of the AGO1­microRNA (AGO1­miRNA) complex, leading to a great attenuation in cytoplasmic AGO1 protein and sRNA levels. With this mechanism, V. dahliae can suppress the accumulation of mobile plant miRNAs in fungal cells and succedent transkingdom silencing of virulence genes, thereby increasing its virulence in plants. Our findings reveal a mechanism by which phytopathogenic fungi antagonize antifungal RNAi-dependent plant immunity and expand the understanding on the complex interaction between host and filamentous pathogens.

Functional analysis of the mating type genes in Verticillium dahliae.

BACKGROUND: Populations of the plant pathogenic fungus Verticillium dahliae display a complex and rich genetic diversity, yet the existence of sexual reproduction in the fungus remains contested. As pivotal genes, MAT genes play a crucial role in regulating cell differentiation, morphological development, and mating of compatible cells. However, the functions of the two mating type genes in V. dahliae, VdMAT1-1-1, and VdMAT1-2-1, remain poorly understood. RESULTS: In this study, we confirmed that the MAT loci in V. dahliae are highly conserved, including both VdMAT1-1-1 and VdMAT1-2-1 which share high collinearity. The conserved core transcription factor encoded by the two MAT loci may facilitate the regulation of pheromone precursor and pheromone receptor genes by directly binding to their promoter regions. Additionally, peptide activity assays demonstrated that the signal peptide of the pheromone VdPpg1 possessed secretory activity, while VdPpg2, lacked a predicted signal peptide. Chemotactic growth assays revealed that V. dahliae senses and grows towards the pheromones FO-a and FO-α of Fusarium oxysporum, as well as towards VdPpg2 of V. dahliae, but not in response to VdPpg1. The findings herein also revealed that VdMAT1-1-1 and VdMAT1-2-1 regulate vegetative growth, carbon source utilization, and resistance to stressors in V. dahliae, while negatively regulating virulence. CONCLUSIONS: These findings underscore the potential roles of VdMAT1-1-1 and VdMAT1-2-1 in sexual reproduction and confirm their involvement in various asexual processes of V. dahliae, offering novel insights into the functions of mating type genes in this species.

Insights into the biocontrol and plant growth promotion functions of Bacillus altitudinis strain KRS010 against Verticillium dahliae.

BACKGROUND: Verticillium wilt, caused by the fungus Verticillium dahliae, is a soil-borne vascular fungal disease, which has caused great losses to cotton yield and quality worldwide. The strain KRS010 was isolated from the seed of Verticillium wilt-resistant Gossypium hirsutum cultivar "Zhongzhimian No. 2." RESULTS: The strain KRS010 has a broad-spectrum antifungal activity to various pathogenic fungi as Verticillium dahliae, Botrytis cinerea, Fusarium spp., Colletotrichum spp., and Magnaporthe oryzae, of which the inhibition rate of V. dahliae mycelial growth was 73.97% and 84.39% respectively through confrontation test and volatile organic compounds (VOCs) treatments. The strain was identified as Bacillus altitudinis by phylogenetic analysis based on complete genome sequences, and the strain physio-biochemical characteristics were detected, including growth-promoting ability and active enzymes. Moreover, the control efficiency of KRS010 against Verticillium wilt of cotton was 93.59%. After treatment with KRS010 culture, the biomass of V. dahliae was reduced. The biomass of V. dahliae in the control group (Vd991 alone) was 30.76-folds higher than that in the treatment group (KRS010+Vd991). From a molecular biological aspect, KRS010 could trigger plant immunity by inducing systemic resistance (ISR) activated by salicylic acid (SA) and jasmonic acid (JA) signaling pathways. Its extracellular metabolites and VOCs inhibited the melanin biosynthesis of V. dahliae. In addition, KRS010 had been characterized as the ability to promote plant growth. CONCLUSIONS: This study indicated that B. altitudinis KRS010 is a beneficial microbe with a potential for controlling Verticillium wilt of cotton, as well as promoting plant growth.

Cotton 4-coumarate-CoA ligase 3 enhanced plant resistance to Verticillium dahliae by promoting jasmonic acid signaling-mediated vascular lignification and metabolic flux.

Lignins and their antimicrobial-related polymers cooperatively enhance plant resistance to pathogens. Several isoforms of 4-coumarate-coenzyme A ligases (4CLs) have been identified as indispensable enzymes involved in lignin and flavonoid biosynthetic pathways. However, their roles in plant-pathogen interaction are still poorly understood. This study uncovers the role of Gh4CL3 in cotton resistance to the vascular pathogen Verticillium dahliae. The cotton 4CL3-CRISPR/Cas9 mutant (CR4cl) exhibited high susceptibility to V. dahliae. This susceptibility was most probably due to the reduction in the total lignin content and the biosynthesis of several phenolic metabolites, e.g., rutin, catechin, scopoletin glucoside, and chlorogenic acid, along with jasmonic acid (JA) attenuation. These changes were coupled with a significant reduction in 4CL activity toward p-coumaric acid substrate, and it is likely that recombinant Gh4CL3 could specifically catalyze p-coumaric acid to form p-coumaroyl-coenzyme A. Thus, overexpression of Gh4CL3 (OE4CL) showed increasing 4CL activity that augmented phenolic precursors, cinnamic, p-coumaric, and sinapic acids, channeling into lignin and flavonoid biosyntheses and enhanced resistance to V. dahliae. Besides, Gh4CL3 overexpression activated JA signaling that instantly stimulated lignin deposition and metabolic flux in response to pathogen, which all established an efficient plant defense response system, and inhibited V. dahliae mycelium growth. Our results propose that Gh4CL3 acts as a positive regulator for cotton resistance against V. dahliae by promoting JA signaling-mediated enhanced cell wall rigidity and metabolic flux.

Cotton peroxisome-localized lysophospholipase counteracts the toxic effects of Verticillium dahliae NLP1 and confers wilt resistance.

Plasma membrane represents a critical battleground between plants and attacking microbes. Necrosis-and-ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs), cytolytic toxins produced by some bacterial, fungal and oomycete species, are able to target on lipid membranes by binding eudicot plant-specific sphingolipids (glycosylinositol phosphorylceramide) and form transient small pores, causing membrane leakage and subsequent cell death. NLP-producing phytopathogens are a big threat to agriculture worldwide. However, whether there are R proteins/enzymes that counteract the toxicity of NLPs in plants remains largely unknown. Here we show that cotton produces a peroxisome-localized enzyme lysophospholipase, GhLPL2. Upon Verticillium dahliae attack, GhLPL2 accumulates on the membrane and binds to V. dahliae secreted NLP, VdNLP1, to block its contribution to virulence. A higher level of lysophospholipase in cells is required to neutralize VdNLP1 toxicity and induce immunity-related genes expression, meanwhile maintaining normal growth of cotton plants, revealing the role of GhLPL2 protein in balancing resistance to V. dahliae and growth. Intriguingly, GhLPL2 silencing cotton plants also display high resistance to V. dahliae, but show severe dwarfing phenotype and developmental defects, suggesting GhLPL2 is an essential gene in cotton. GhLPL2 silencing results in lysophosphatidylinositol over-accumulation and decreased glycometabolism, leading to a lack of carbon sources required for plants and pathogens to survive. Furthermore, lysophospholipases from several other crops also interact with VdNLP1, implying that blocking NLP virulence by lysophospholipase may be a common strategy in plants. Our work demonstrates that overexpressing lysophospholipase encoding genes have great potential for breeding crops with high resistance against NLP-producing microbial pathogens.

Acetylation of GhCaM7 enhances cotton resistance to Verticillium dahliae.

Protein lysine acetylation is an important post-translational modification mechanism involved in cellular regulation in eukaryotes. Calmodulin (CaM) is a ubiquitous Ca2+ sensor in eukaryotes and is crucial for plant immunity, but it is so far unclear whether acetylation is involved in CaM-mediated plant immunity. Here, we found that GhCaM7 is acetylated upon Verticillium dahliae (V. dahliae) infection and a positive regulator of V. dahliae resistance. Overexpressing GhCaM7 in cotton and Arabidopsis enhances V. dahliae resistance and knocking-down GhCaM7 makes cotton more susceptible to V. dahliae. Transgenic Arabidopsis plants overexpressing GhCaM7 with mutation at the acetylation site are more susceptible to V. dahliae than transgenics overexpressing the wild-type GhCaM7, implying the importance of the acetylated GhCaM7 in response to V. dahliae infection. Yeast two-hybrid, bimolecular fluorescent complementation, luciferase complementation imaging, and coimmunoprecipitation assays demonstrated interaction between GhCaM7 and an osmotin protein GhOSM34 that was shown to have a positive role in V. dahliae resistance. GhCaM7 and GhOSM34 are co-localized in the cell membrane. Upon V. dahliae infection, the Ca2+ content reduces almost instantly in plants with downregulated GhCaM7 or GhOSM34. Down regulating GhOSM34 enhances accumulation of Na+ and increases cell osmotic pressure. Comparative transcriptomic analyses between cotton plants with an increased or reduced expression level of GhCaM7 and wild-type plants indicate the involvement of jasmonic acid signaling pathways and reactive oxygen species in GhCaM7-enabled disease resistance. Together, these results demonstrate the involvement of CaM protein in the interaction between cotton and V. dahliae, and more importantly, the involvement of the acetylated CaM in the interaction.

The ScAPD1-like gene from the desert moss Syntrichia caninervis enhances resistance to Verticillium dahliae via phenylpropanoid gene regulation.

Soloist is a member of a distinct and small subfamily within the AP2/ERF transcriptional factor family that play important roles in plant biotic and abiotic stress responses. There are limited studies of Soloist genes and their functions are poorly understood. We characterized the abiotic and biotic stress tolerance function of the ScSoloist gene (designated as ScAPD1-like) from the desert moss Syntrichia caninervis. ScAPD1-like responded to multiple abiotic, biotic stresses and plant hormone treatments. ScAPD1-like protein located to the nucleus and bound to several DNA elements. Overexpression of ScAPD1-like in Arabidopsis did not alter abiotic stress resistance or inhibit Pseudomonas syringae pv. tomato (Pst) DC3000 infection. However, overexpression of ScAPD1-like significantly increased the resistance of transgenic Arabidopsis and S. caninervis to Verticillium dahliae infection, decreased reactive oxygen species accumulation and improved reactive oxygen species scavenging activity. ScAPD1-like overexpression plants altered the abundance of transcripts for lignin synthesis and promoted lignin accumulation in Arabidopsis. ScAPD1-like directly bind to RAV1, AC elements, and TATA-box in the promoters of AtPAL1 and AtC4H genes, respectively, in vitro. Chromatin immunoprecipitation-quantitative polymerase chain reaction assays demonstrated ScAPD1-like directly bound to PAL and C4H genes promoters in Arabidopsis and their homologs in S. caninervis. In S. caninervis, ScAPD1-like overexpression and RNAi directly regulated the abundance of ScPAL and ScC4H transcripts and modified the metabolites of phenylpropanoid pathway. We provide insight into the function of Soloist in plant defense mechanisms that likely occurs through activation of the phenylpropanoid biosynthesis pathway. ScAPD1-like is a promising candidate gene for breeding strategies to improve resistance to Verticillium wilt.

The Verticillium dahliae Effector VdPHB1 Promotes Pathogenicity in Cotton and Interacts with the Immune Protein GhMC4.

Verticillium dahliae is a kind of pathogenic fungus that brings about wilt disease and great losses in cotton. The molecular mechanism of the effectors in V. dahliae regulating cotton immunity remains largely unknown. Here, we identified an effector of V. dahliae, VdPHB1, whose gene expression is highly induced by infection. The VdPHB1 protein is localized to the intercellular space of cotton plants. Knock-out of the VdPHB1 gene in V. dahliae had no effect on pathogen growth, but decreased the virulence in cotton. VdPHB1 ectopically expressed Arabidopsis plants were growth-inhibited and significantly susceptible to V. dahliae. Further, VdPHB1 interacted with the type II metacaspase GhMC4. GhMC4 gene-silenced cotton plants were more sensitive to V. dahliae with reduced expression of pathogen defense-related and programmed cell death genes. The accumulation of GhMC4 protein was concurrently repressed when VdPHB1 protein was expressed during infection. In summary, these results have revealed a novel molecular mechanism of virulence regulation that the secreted effector VdPHB1 represses the activity of cysteine protease for helping V. dahliae infection in cotton.

Analysis of endophytic bacterial diversity in seeds of different genotypes of cotton and the suppression of Verticillium wilt pathogen infection by a synthetic microbial community.

BACKGROUND: In agricultural production, fungal diseases significantly impact the yield and quality of cotton (Gossypium spp.) with Verticillium wilt posing a particularly severe threat. RESULTS: This study is focused on investigating the effectiveness of endophytic microbial communities present in the seeds of disease-resistant cotton genotypes in the control of cotton Verticillium wilt. The technique of 16S ribosomal RNA (16S rRNA) amplicon sequencing identified a significant enrichment of the Bacillus genus in the resistant genotype Xinluzao 78, which differed from the endophytic bacterial community structure in the susceptible genotype Xinluzao 63. Specific enriched strains were isolated and screened from the seeds of Xinluzao 78 to further explore the biological functions of seed endophytes. A synthetic microbial community (SynCom) was constructed using the broken-rod model, and seeds of the susceptible genotype Xinluzao 63 in this community that had been soaked with the SynCom were found to significantly control the occurrence of Verticillium wilt and regulate the growth of cotton plants. Antibiotic screening techniques were used to preliminarily identify the colonization of strains in the community. These techniques revealed that the strains can colonize plant tissues and occupy ecological niches in cotton tissues through a priority effect, which prevents infection by pathogens. CONCLUSION: This study highlights the key role of seed endophytes in driving plant disease defense and provides a theoretical basis for the future application of SynComs in agriculture.

Transcriptome analysis of Gossypium hirsutum cultivar Zhongzhimian No.2 uncovers the gene regulatory networks involved in defense against Verticillium dahliae.

BACKGROUND: Cotton is globally important crop. Verticillium wilt (VW), caused by Verticillium dahliae, is the most destructive disease in cotton, reducing yield and fiber quality by over 50% of cotton acreage. Breeding resistant cotton cultivars has proven to be an efficient strategy for improving the resistance of cotton to V. dahliae. However, the lack of understanding of the genetic basis of VW resistance may hinder the progress in deploying elite cultivars with proven resistance. RESULTS: We planted the VW-resistant Gossypium hirsutum cultivar Zhongzhimian No.2 (ZZM2) in an artificial greenhouse and disease nursery. ZZM2 cotton was subsequently subjected to transcriptome sequencing after Vd991 inoculation (6, 12, 24, 48, and 72 h post-inoculation). Several differentially expressed genes (DEGs) were identified in response to V. dahliae infection, mainly involved in resistance processes, such as flavonoid and terpenoid quinone biosynthesis, plant hormone signaling, MAPK signaling, phenylpropanoid biosynthesis, and pyruvate metabolism. Compared to the susceptible cultivar Junmian No.1 (J1), oxidoreductase activity and reactive oxygen species (ROS) production were significantly increased in ZZM2. Furthermore, gene silencing of cytochrome c oxidase subunit 1 (COX1), which is involved in the oxidation-reduction process in ZZM2, compromised its resistance to V. dahliae, suggesting that COX1 contributes to VW resistance in ZZM2. CONCLUSIONS: Our data demonstrate that the G. hirsutum cultivar ZZM2 responds to V. dahliae inoculation through resistance-related processes, especially the oxidation-reduction process. This enhances our understanding of the mechanisms regulating the ZZM2 defense against VW.

The elicitor VP2 from Verticillium dahliae triggers defence response in cotton.

Verticillium dahliae is a widespread and destructive soilborne vascular pathogenic fungus that causes serious diseases in dicot plants. Here, comparative transcriptome analysis showed that the number of genes upregulated in defoliating pathotype V991 was significantly higher than in the non-defoliating pathotype 1cd3-2 during the early response of cotton. Combined with analysis of the secretome during the V991-cotton interaction, an elicitor VP2 was identified, which was highly upregulated at the early stage of V991 invasion, but was barely expressed during the 1cd3-2-cotton interaction. Full-length VP2 could induce cell death in several plant species, and which was dependent on NbBAK1 but not on NbSOBIR1 in N. benthamiana. Knock-out of VP2 attenuated the pathogenicity of V991. Furthermore, overexpression of VP2 in cotton enhanced resistance to V. dahliae without causing abnormal plant growth and development. Several genes involved in JA, SA and lignin synthesis were significantly upregulated in VP2-overexpressing cotton. The contents of JA, SA, and lignin were also significantly higher than in the wild-type control. In summary, the identified elicitor VP2, recognized by the receptor in the plant membrane, triggers the cotton immune response and enhances disease resistance.

The interplay of suppressive soil bacteria and plant root exudates determines germination of microsclerotia of Verticillium longisporum.

Dormant microsclerotia play a vital role in the survival and spread of Verticillium longisporum, as they can stay viable in the soil and maintain their infectivity for many years. In our previous work, we revealed that soil bacterial volatiles are a key inhibitory factor causing microsclerotia dormancy in the soil. In this study, we further demonstrate that root exudates collected from both host and non-host plants can effectively rescue microsclerotia from bacterial suppression and initiate germination. To identify the specific compounds in root exudates responsible for microsclerotia germination, we fractionated the collected root exudates into polar and non-polar compounds. Subsequently, we conducted comprehensive bioassays with each fraction on germination-suppressed microsclerotia. The result revealed a pivotal role of primary metabolites in root exudates, particularly glutamic acid, in triggering microsclerotia germination and overcoming bacterial inhibition. Moreover, our studies revealed a decrease in inhibitory bacterial volatile fatty acids when bacteria were cultured in the presence of root exudates or glutamic acid. This suggests a potential mechanism, by which root exudates set-off bacterial suppression on microsclerotia. Here, we reveal for the first time that plant root exudates, instead of directly inducing the germination of microsclerotia, enact a set-off effect by counteracting the suppressive impact of soil bacteria on the microsclerotia germination process. This nuanced interaction advances our understanding of the multifaceted dynamics governing microsclerotia dormancy and germination in the soil environment. IMPORTANCE: Our research provides first-time insights into the crucial interaction between plant root exudates and soil bacteria in regulating the germination of Verticillium longisporum microsclerotia, a significant structure in the survival and proliferation of this soil-borne pathogen. We describe so far unknown mechanisms, which are key to understand how root infections on oilseed rape can occur. By pinpointing primary metabolites in root exudates as key factors in overcoming bacteria-induced dormancy and promote microsclerotia germination, our study highlights the potential for exploiting plant - as well as soil microbe-derived - compounds to control V. longisporum. This work underscores the importance of elucidating the nuanced interactions within the soil ecosystem to devise innovative strategies for managing root infective plant diseases, thereby contributing to the resilience and health of cropping systems.

Strigolactones positively regulate Verticillium wilt resistance in cotton via crosstalk with other hormones.

Verticillium wilt caused by Verticillium dahliae is a serious vascular disease in cotton (Gossypium spp.). V. dahliae induces the expression of the CAROTENOID CLEAVAGE DIOXYGENASE 7 (GauCCD7) gene involved in strigolactone (SL) biosynthesis in Gossypium australe, suggesting a role for SLs in Verticillium wilt resistance. We found that the SL analog rac-GR24 enhanced while the SL biosynthesis inhibitor TIS108 decreased cotton resistance to Verticillium wilt. Knock-down of GbCCD7 and GbCCD8b genes in island cotton (Gossypium barbadense) decreased resistance, whereas overexpression of GbCCD8b in upland cotton (Gossypium hirsutum) increased resistance to Verticillium wilt. Additionally, Arabidopsis (Arabidopsis thaliana) SL mutants defective in CCD7 and CCD8 putative orthologs were susceptible, whereas both Arabidopsis GbCCD7- and GbCCD8b-overexpressing plants were more resistant to Verticillium wilt than wild-type (WT) plants. Transcriptome analyses showed that several genes related to the jasmonic acid (JA)- and abscisic acid (ABA)-signaling pathways, such as MYELOCYTOMATOSIS 2 (GbMYC2) and ABA-INSENSITIVE 5, respectively, were upregulated in the roots of WT cotton plants in responses to rac-GR24 and V. dahliae infection but downregulated in the roots of both GbCCD7- and GbCCD8b-silenced cotton plants. Furthermore, GbMYC2 suppressed the expression of GbCCD7 and GbCCD8b by binding to their promoters, which might regulate the homeostasis of SLs in cotton through a negative feedback loop. We also found that GbCCD7- and GbCCD8b-silenced cotton plants were impaired in V. dahliae-induced reactive oxygen species (ROS) accumulation. Taken together, our results suggest that SLs positively regulate cotton resistance to Verticillium wilt through crosstalk with the JA- and ABA-signaling pathways and by inducing ROS accumulation.

VILLIN2 regulates cotton defense against Verticillium dahliae by modulating actin cytoskeleton remodeling.

The active structural change of actin cytoskeleton is a general host response upon pathogen attack. This study characterized the function of the cotton (Gossypium hirsutum) actin-binding protein VILLIN2 (GhVLN2) in host defense against the soilborne fungus Verticillium dahliae. Biochemical analysis demonstrated that GhVLN2 possessed actin-binding, -bundling, and -severing activities. A low concentration of GhVLN2 could shift its activity from actin bundling to actin severing in the presence of Ca2+. Knockdown of GhVLN2 expression by virus-induced gene silencing reduced the extent of actin filament bundling and interfered with the growth of cotton plants, resulting in the formation of twisted organs and brittle stems with a decreased cellulose content of the cell wall. Upon V. dahliae infection, the expression of GhVLN2 was downregulated in root cells, and silencing of GhVLN2 enhanced the disease tolerance of cotton plants. The actin bundles were less abundant in root cells of GhVLN2-silenced plants than in control plants. However, upon infection by V. dahliae, the number of actin filaments and bundles in the cells of GhVLN2-silenced plants was raised to a comparable level as those in control plants, with the dynamic remodeling of the actin cytoskeleton appearing several hours in advance. GhVLN2-silenced plants exhibited a higher incidence of actin filament cleavage in the presence of Ca2+, suggesting that pathogen-responsive downregulation of GhVLN2 could activate its actin-severing activity. These data indicate that the regulated expression and functional shift of GhVLN2 contribute to modulating the dynamic remodeling of the actin cytoskeleton in host immune responses against V. dahliae.

General Regulatory Factor7 regulates innate immune signalling to enhance Verticillium wilt resistance in cotton.

Sessile growing plants are always vulnerable to microbial pathogen attacks throughout their lives. To fend off pathogen invasion, plants have evolved a sophisticated innate immune system that consists of cell surface receptors and intracellular receptors. Somatic embryogenesis receptor kinases (SERKs) belong to a small group of leucine-rich repeat receptor-like kinases (LRR-RLKs) that function as co-receptors regulating diverse physiological processes. GENRAL REGULATORY FACTOR (GRF) proteins play an important role in physiological signalling transduction. However, the function of GRF proteins in plant innate immune signalling remains elusive. Here, we identified a GRF gene, GauGRF7, that is expressed both constitutively and in response to fungal pathogen infection. Intriguingly, silencing of GRF7 compromised plant innate immunity, resulting in susceptibility to Verticillium dahliae infection. Both transgenic GauGRF7 cotton and transgenic GauGRF7 Arabidopsis lines enhanced the innate immune response to V. dahliae infection, leading to high expression of two helper NLRs (hNLR) genes (ADR1 and NRG1) and pathogenesis-related genes, and increased ROS production and salicylic acid level. Moreover, GauGRF7 interacted with GhSERK1, which positively regulated GRF7-mediated innate immune response in cotton and Arabidopsis. Our findings revealed the molecular mechanism of the GRF protein in plant immune signaling and offer potential opportunities for improving plant resistance to V. dahliae infection.