Exploring the biocontrol potential of rocket (Eruca sativa) extracts and associated microorganisms against Verticillium wilt.

AIMS: This study aimed to assess the impact of rocket (Eruca sativa) extract on Verticillium wilt in eggplants, explore rhizospheric microorganisms for disease biocontrol, and evaluate selected strains' induced systemic resistance (ISR) potential while characterizing their genomic and biosynthetic profiles. METHODS AND RESULTS: Rocket extract application led to a significant reduction in Verticillium wilt symptoms in eggplants compared to controls. Isolated microorganisms from treated soil, including Paraburkholderia oxyphila EP1, Pseudomonas citronellolis EP2, Paraburkholderia eburnea EP3, and P. oxyphila EP4 and EP5, displayed efficacy against Verticillium dahliae, decreasing disease severity and incidence in planta. Notably, strains EP3 and EP4 triggered ISR in eggplants against V. dahliae. Genomic analysis unveiled shared biosynthetic gene clusters, such as ranthipeptide and non-ribosomal peptide synthetase-metallophore types, among the isolated strains. Additionally, metabolomic profiling of EP2 revealed the production of metabolites associated with amino acid metabolism, putative antibiotics, and phytohormones. CONCLUSIONS: The application of rocket extract resulted in a significant reduction in Verticillium wilt symptoms in eggplants, while the isolated microorganisms displayed efficacy against V. dahliae, inducing systemic resistance and revealing shared biosynthetic gene clusters, with metabolomic profiling highlighting potential disease-suppressing metabolites.

A systematic analysis of ARM genes revealed that GhARM144 regulates the resistance against Verticillium dahliae via interaction with GhOSM34.

Proteins of the armadillo repeat gene family play important roles in plant pathogen response. Here, 169 armadillo (ARM) genes were identified in upland cotton (Gossypium hirsutum). Phylogenetic analysis grouped these into 11 subfamilies, with conserved protein structures within each subfamily. The results signify that the expansion of the gene family occurred via whole genome duplication and dispersed duplication. Expression profiling and network analysis suggest that GhARM144 may regulate cotton resistance to Verticillium dahliae. GhARM144 was upregulated in roots by V. dahliae infection or salicylic acid treatment. This upregulation indicates a negative regulatory role of GhARM144' in the cotton immune responses, potentially by manipulating salicylic acid biosynthesis. Protein interaction studies found that GhARM144 associates with an osmotin-like protein, GhOSM34, at the plasma membrane. Silencing GhOSM34 reduced the resistance to V. dahliae, suggesting it may play a positive regulatory role. The results demonstrate that GhARM144 modulates cotton immunity through interaction with GhOSM34 and salicylic acid signalling. Further study of these proteins may yield insights into disease resistance mechanisms in cotton and other plants.

StoMYB41 positively regulates the Solanum torvum response to Verticillium dahliae in an ABA dependent manner.

MYB transcription factor despite their solid involvement in growth are potent regulator of plant stress response. Herein, we identified a MYB gene named as StoMYB41 in a wild eggplant species Solanum torvum. The expression level of StoMYB41 was higher in root than the tissues including stem, leaf, and seed. It induced significantly by Verticillium dahliae inoculation. StoMYB41 was localized in the nucleus and exhibited transcriptional activation activity. Silencing of StoMYB41 enhanced susceptibility of Solanum torvum against Verticillium dahliae, accompanied by higher disease index. The significant down-regulation of resistance marker gene StoABR1 comparing to the control plants was recorded in the silenced plants. Moreover, transient expression of StoMYB41 could trigger intense hypersensitive reaction mimic cell death, darker DAB and trypan blue staining, higher ion leakage, and induced the expression levels of StoABR1 and NbDEF1 in the leaves of Solanum torvum and Nicotiana benthamiana. Taken together, our data indicate that StoMYB41 acts as a positive regulator in Solanum torvum against Verticillium wilt.

Hen Egg White Lysozyme (HEWL) Confers Resistance to Verticillium Wilt in Cotton by Inhibiting the Spread of Fungus and Generating ROS Burst.

Verticillium wilt is a soil-borne vascular disease caused by the fungal pathogen Verticillium dahliae. It causes great harm to upland cotton (Gossypium hirsutum) yield and quality. A previous study has shown that Hen egg white lysozyme (HEWL) exerts strong inhibitory activity against V. dahliae in vitro. In the current study, we introduced the HEWL gene into cotton through the Agrobacterium-mediated transformation, and the exogenous HEWL protein was successfully expressed in cotton. Our study revealed that HEWL was able to significantly inhibit the proliferation of V. dahlia in cotton. Consequently, the overexpression of HEWL effectively improved the resistance to Verticillium wilt in transgenic cotton. In addition, ROS accumulation and NO content increased rapidly after the V. dahliae inoculation of plant leaves overexpressing HEWL. In addition, the expression of the PR genes was significantly up-regulated. Taken together, our results suggest that HEWL significantly improves resistance to Verticillium wilt by inhibiting the growth of pathogenic fungus, triggering ROS burst, and activating PR genes expression in cotton.

Decoding the guardians of cotton resilience: A comprehensive exploration of the ßCA genes and its role in Verticillium dahliae resistance.

Plant Carbonic anhydrases (Cas) have been shown to be stress-responsive enzymes that may play a role in adapting to adverse conditions. Cotton is a significant economic crop in China, with upland cotton (Gossypium hirsutum) being the most widely cultivated species. We conducted genome-wide identification of the ßCA gene in six cotton species and preliminary analysis of the ßCA gene in upland cotton. In total, 73 ßCA genes from six cotton species were identified, with phylogenetic analysis dividing them into five subgroups. GHßCA proteins were predominantly localized in the chloroplast and cytoplasm. The genes exhibited conserved motifs, with motifs 1, 2, and 3 being prominent. GHßCA genes were unevenly distributed across chromosomes and were associated with stress-responsive cis-regulatory elements, including those responding to light, MeJA, salicylic acid, abscisic acid, cell cycle regulation, and defence/stress. Expression analysis indicated that GHßCA6, GHßCA7, GHßCA10, GHßCA15, and GHßCA16 were highly expressed under various abiotic stress conditions, whereas GHßCA3, GHßCA9, GHßCA10, and GHßCA18 had higher expression patterns under Verticillium dahliae infection at different time intervals. In Gossypium thurberi, GthßCA1, GthßCA2, and GthßCA4 showed elevated expression across stress conditions and tissues. Silencing GHßCA10 through VIGS increased Verticillium wilt severity and reduced lignin deposition compared to non-silenced plants. GHßCA10 is crucial for cotton's defense against Verticillium dahliae. Further research is needed to understand the underlying mechanisms and develop strategies to enhance resistance against Verticillium wilt.

GhTLP1, a thaumatin-like protein 1, improves Verticillium wilt resistance in cotton via JA, ABA and MAPK signaling pathway-plant pathways.

Verticillium wilt of cotton is a very serious soil-borne disease and there is no effective control method. The mechanism of Gossypium hirsutum thaumatin-like protein 1(GhTLP1) in upland cotton regulating Verticillium wilt resistance has been an uncovered research approach. GhTLP1 is mainly localized in the cell wall. Overexpression of GhTLP1 significantly enhanced Arabidopsis plants resistance to Verticillium dahliae, while its homologous mutant tlp1 in Arabidopsis was more susceptible to the pathogen, and the heterologous complement line (EC) recovered resistance to V. dahliae. GhTLP1 responds to jasmonate acid (JA) and abscisic acid (ABA) hormones and regulates mitogen-activated protein kinase (MAPK) signaling pathway-plant pathway to enhance Arabidopsis plants resistance to V. dahliae. Silencing GhTLP1 resulted decrease in cotton plants resistance to V. dahliae. Moreover, the mutation of GhTLP1 at site Tyr97 and Tyr199 with the phosphorylation also decreased plant resistance to V. dahliae. Therefore, GhTLP1 phosphorylation was observed important in cotton plants against V. dahliae. Further analysis demonstrated that GhTLP1 interacted with gossypium hirsutum laccase 14 (GhLAC14) to enhance plants resistance to V. dahliae. Silencing GhLAC14 resulted decrease in cotton plants resistance to V. dahliae. Here, we propose that GhTLP1 is a potential molecular target for improving resistance to Verticillium wilt in cotton.

Genome-wide identification and analysis of a cotton secretome reveals its role in resistance against Verticillium dahliae.

BACKGROUND: The extracellular space between the cell wall and plasma membrane is a battlefield in plant-pathogen interactions. Within this space, the pathogen employs its secretome to attack the host in a variety of ways, including immunity manipulation. However, the role of the plant secretome is rarely studied for its role in disease resistance. RESULTS: Here, we examined the secretome of Verticillium wilt-resistant Gossypium hirsutum cultivar Zhongzhimian No.2 (ZZM2, encoding 95,327 predicted coding sequences) to determine its role in disease resistance against the wilt causal agent, Verticillium dahliae. Bioinformatics-driven analyses showed that the ZZM2 genome encodes 2085 secreted proteins and that these display disequilibrium in their distribution among the chromosomes. The cotton secretome displayed differences in the abundance of certain amino acid residues as compared to the remaining encoded proteins due to the localization of these putative proteins in the extracellular space. The secretome analysis revealed conservation for an allotetraploid genome, which nevertheless exhibited variation among orthologs and comparable unique genes between the two sub-genomes. Secretome annotation strongly suggested its involvement in extracellular stress responses (hydrolase activity, oxidoreductase activity, and extracellular region, etc.), thus contributing to resistance against the V. dahliae infection. Furthermore, the defense response genes (immunity marker NbHIN1, salicylic acid marker NbPR1, and jasmonic acid marker NbLOX4) were activated to varying degrees when Nicotina benthamiana leaves were agro-infiltrated with 28 randomly selected members, suggesting that the secretome plays an important role in the immunity response. Finally, gene silencing assays of 11 members from 13 selected candidates in ZZM2 displayed higher susceptibility to V. dahliae, suggesting that the secretome members confer the Verticillium wilt resistance in cotton. CONCLUSIONS: Our data demonstrate that the cotton secretome plays an important role in Verticillium wilt resistance, facilitating the development of the resistance gene markers and increasing the understanding of the mechanisms regulating disease resistance.

Glycolate oxidase gene family identification and functional analyses in cotton resistance to Verticillium wilt.

Glycolate oxidase (GOX) plays an important role in the regulation of photorespiration and photosynthesis in plants. However, as one of the main enzymes to produce the second messenger hydrogen peroxide (H2O2), its functions in response to pathogens are still poorly understood. In this study, we carried out genome-wide identification, and 14 GOX genes were identified in Gossypium hirsutum. These GOX genes are located on 10 chromosomes and divided into hydroxyacid-oxidases (HAOX) and GOX groups. After infection with Verticillium dahliae Kleb., six GOX gene expression levels were changed. Moreover, H2O2, salicylic acid (SA), and the content and activity of GOX increased in cotton. GhHAOX2-D-silenced plants showed more wilting than control plants after infection with V. dahliae. Additionally, H2O2 accumulation and SA content were reduced in GhHAOX2-D-silenced plants. The expression levels of GhPAL, GhPAD4, and GhPR1 and the lignin content of the silenced plants were significantly lower than those of the control plants. These results indicate that GhHAOX2-D is a positive regulator of Verticillium wilt tolerance in cotton and may promote H2O2 accumulation via the synergistic effects of GOX genes and SA. Collectively, GOX genes play an important role in cotton resistance to Verticillium wilt.

Genome-wide identification of xylan glucuronosyltransferase family in cotton and function characterization of GhGUX5 in regulating Verticillium wilt resistance.

Xylan glucuronosyltransferase (GUX) is widely involved in a variety of physiological processes in plants, including plant development, growth and the defense response to pathogens. However, the function of GUX regulators in Verticillium dahliae (V. dahliae) infection has not been considered previously in cotton. Overall, 119 GUX genes were identified from multiple species and were phylogenetically categorized into seven classes. Duplication event analysis indicated that GUXs in Gossypium hirsutum primarily originated from segmental duplication. GhGUXs promoter analysis indicated cis-regulatory elements capable of reacting to several different stresses. RNA-Seq data and qRT-PCR analysis both indicated that most GhGUXs were associated with V. dahliae infection. Gene interaction network analysis showed that GhGUX5 interacted with 11 proteins, and the relative expression of these 11 proteins changed significantly following V. dahliae infection. In addition, silencing and overexpression of GhGUX5 results to enhance and reduce plant's susceptibility to V. dahliae. Further study showed that TRV:GhGUX5 silenced cotton plants exhibited a decrease in the degree of lignification, total lignin content, gene expression levels involved in lignin biosynthesis, and enzyme activity compared with TRV:00. The above results indicate that GhGUX5 enhances Verticillium wilt resistance through the lignin biosynthesis pathway.

Insights to Gossypium defense response against Verticillium dahliae: the Cotton Cancer.

The soil-borne pathogen Verticillium dahliae, also referred as "The Cotton Cancer," is responsible for causing Verticillium wilt in cotton crops, a destructive disease with a global impact. To infect cotton plants, the pathogen employs multiple virulence mechanisms such as releasing enzymes that degrade cell walls, activating genes that contribute to virulence, and using protein effectors. Conversely, cotton plants have developed numerous defense mechanisms to combat the impact of V. dahliae. These include strengthening the cell wall by producing lignin and depositing callose, discharging reactive oxygen species, and amassing hormones related to defense. Despite the efforts to develop resistant cultivars, there is still no permanent solution to Verticillium wilt due to a limited understanding of the underlying molecular mechanisms that drive both resistance and pathogenesis is currently prevalent. To address this challenge, cutting-edge technologies such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), host-induced gene silencing (HIGS), and gene delivery via nano-carriers could be employed as effective alternatives to control the disease. This article intends to present an overview of V. dahliae virulence mechanisms and discuss the different cotton defense mechanisms against Verticillium wilt, including morphophysiological and biochemical responses and signaling pathways including jasmonic acid (JA), salicylic acid (SA), ethylene (ET), and strigolactones (SLs). Additionally, the article highlights the significance of microRNAs (miRNAs), circular RNAs (circRNAs), and long non-coding RNAs (lncRNAs) in gene expression regulation, as well as the different methods employed to identify and functionally validate genes to achieve resistance against this disease. Gaining a more profound understanding of these mechanisms could potentially result in the creation of more efficient strategies for combating Verticillium wilt in cotton crops.

Effect of volatile compounds produced by the cotton endophytic bacterial strain Bacillus sp. T6 against Verticillium wilt.

BACKGROUND: Verticillium wilt, caused by the fungus Verticillium dahliae, leads to significant losses in cotton yield worldwide. Biocontrol management is a promising means of suppressing verticillium wilt. The purpose of the study was to obtain and analyze endophytic bacteria with Verticillium wilt-resistant activities from the roots of Gossypium barbadense 'Xinhai15' and to explore the interactions between the soil and plants. RESULTS: An endophytic bacterium Bacillus sp. T6 was obtained from the Verticillium wilt-resistant cotton G. barbadense 'Xinhai15', which showed significant antagonistic abilities against cotton Verticillium wilt. The bioassay results indicated that the strain possessed strong antagonistic abilities that inhibited V. dahliae spore germination and mycelial growth without contact, and thus it was speculated that the active factor of the bacteria might be volatile compounds. A total of 46 volatile substances were detected via headspace solid-phase microextraction and gas chromatography-mass spectrometry analysis. The pure product verification experiment confirmed that the styrene produced by the T6 strain was the main virulence factor. Transcriptome analysis showed that following styrene induction, 247 genes in V. dahliae, including four hydrolase genes, eight dehydrogenase genes, 11 reductase genes, 17 genes related to transport and transfer were upregulated. Additionally, 72 genes, including two chitinase genes, two protease genes, five transport-related genes, and 33 hypothetical protein genes, were downregulated. The quantitative real-time PCR results confirmed that the expression of the four genes VDAG_02838, VDAG_09554, VDAG_045572, and VDAG_08251 was increased by 3.18, 78.83, 2.71, and 2.92 times, respectively, compared with the uninduced control group. CONCLUSIONS: The research provides a new reference for the development and application of the volatile compounds of endophytic bacteria as new biocontrol agents for the control of Verticillium wilt and as biological preservatives for agricultural products.

A polyketide synthase from Verticillium dahliae modulates melanin biosynthesis and hyphal growth to promote virulence.

BACKGROUND: During the disease cycle, plant pathogenic fungi exhibit a morphological transition between hyphal growth (the phase of active infection) and the production of long-term survival structures that remain dormant during "overwintering." Verticillium dahliae is a major plant pathogen that produces heavily melanized microsclerotia (MS) that survive in the soil for 14 or more years. These MS are multicellular structures produced during the necrotrophic phase of the disease cycle. Polyketide synthases (PKSs) are responsible for catalyzing production of many secondary metabolites including melanin. While MS contribute to long-term survival, hyphal growth is key for infection and virulence, but the signaling mechanisms by which the pathogen maintains hyphal growth are unclear. RESULTS: We analyzed the VdPKSs that contain at least one conserved domain potentially involved in secondary metabolism (SM), and screened the effect of VdPKS deletions in the virulent strain AT13. Among the five VdPKSs whose deletion affected virulence on cotton, we found that VdPKS9 acted epistatically to the VdPKS1-associated melanin pathway to promote hyphal growth. The decreased hyphal growth in VdPKS9 mutants was accompanied by the up-regulation of melanin biosynthesis and MS formation. Overexpression of VdPKS9 transformed melanized hyphal-type (MH-type) into the albinistic hyaline hyphal-type (AH-type), and VdPKS9 was upregulated in the AH-type population, which also exhibited higher virulence than the MH-type. CONCLUSIONS: We show that VdPKS9 is a powerful negative regulator of both melanin biosynthesis and MS formation in V. dahliae. These findings provide insight into the mechanism of how plant pathogens promote their virulence by the maintenance of vegetative hyphal growth during infection and colonization of plant hosts, and may provide novel targets for the control of melanin-producing filamentous fungi.

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.

Verticillium dahliae CFEM proteins manipulate host immunity and differentially contribute to virulence.

BACKGROUND: Verticillium dahliae is a fungal pathogen that causes a vascular wilt on many economically important crops. Common fungal extracellular membrane (CFEM) domain proteins including secreted types have been implicated in virulence, but their roles in this pathogen are still unknown. RESULTS: Nine secreted small cysteine-rich proteins (VdSCPs) with CFEM domains were identified by bioinformatic analyses and their differential suppression of host immune responses were evaluated. Two of these proteins, VdSCP76 and VdSCP77, localized to the plant plasma membrane owing to their signal peptides and mediated broad-spectrum suppression of all immune responses induced by typical effectors. Deletion of either VdSCP76 or VdSCP77 significantly reduced the virulence of V. dahliae on cotton. Furthermore, VdSCP76 and VdSCP77 suppressed host immunity through the potential iron binding site conserved in CFEM family members, characterized by an aspartic acid residue in seven VdSCPs (Asp-type) in contrast with an asparagine residue (Asn-type) in VdSCP76 and VdSCP77. V. dahliae isolates carrying the Asn-type CFEM members were more virulent on cotton than those carrying the Asp-type. CONCLUSIONS: In the iron-insufficient xylem, V. dahliae is likely to employ the Asp-type CFEM members to chelate iron, and Asn-type CFEM members to suppress immunity, for successful colonization and propagation in host plants.

An ancient antimicrobial protein co-opted by a fungal plant pathogen for in planta mycobiome manipulation.

Microbes typically secrete a plethora of molecules to promote niche colonization. Soil-dwelling microbes are well-known producers of antimicrobials that are exploited to outcompete microbial coinhabitants. Also, plant pathogenic microbes secrete a diversity of molecules into their environment for niche establishment. Upon plant colonization, microbial pathogens secrete so-called effector proteins that promote disease development. While such effectors are typically considered to exclusively act through direct host manipulation, we recently reported that the soil-borne, fungal, xylem-colonizing vascular wilt pathogen Verticillium dahliae exploits effector proteins with antibacterial properties to promote host colonization through the manipulation of beneficial host microbiota. Since fungal evolution preceded land plant evolution, we now speculate that a subset of the pathogen effectors involved in host microbiota manipulation evolved from ancient antimicrobial proteins of terrestrial fungal ancestors that served in microbial competition prior to the evolution of plant pathogenicity. Here, we show that V. dahliae has co-opted an ancient antimicrobial protein as effector, named VdAMP3, for mycobiome manipulation in planta. We show that VdAMP3 is specifically expressed to ward off fungal niche competitors during resting structure formation in senescing mesophyll tissues. Our findings indicate that effector-mediated microbiome manipulation by plant pathogenic microbes extends beyond bacteria and also concerns eukaryotic members of the plant microbiome. Finally, we demonstrate that fungal pathogens can exploit plant microbiome-manipulating effectors in a life stage-specific manner and that a subset of these effectors has evolved from ancient antimicrobial proteins of fungal ancestors that likely originally functioned in manipulation of terrestrial biota.

The Cotton Lignin Biosynthetic Gene Gh4CL30 Regulates Lignification and Phenolic Content and Contributes to Verticillium Wilt Resistance.

Verticillium wilt is a vascular disease causing tremendous damage to cotton production worldwide. However, our knowledge of the mechanisms of cotton resistance or susceptibility to this disease is very limited. In this study, we compared the defense transcriptomes of cotton (Gossypium hirsutum) cultivars Shidalukang 1 (Verticillium dahliae resistant, HR) and Junmian 1 (V. dahliae susceptible, HS) before and after V. dahliae infection, identified hub genes of the network associated with responses to V. dahliae infection, and functionally characterized one of the hub genes involved in biosynthesis of lignin and phenolics. We identified 6,831 differentially expressed genes (DEGs) between the basal transcriptomes of HR and HS; 3,685 and 3,239 of these DEGs were induced in HR and HS, respectively, at different time points after V. dahliae infection. KEGG pathway analysis indicated that DEGs were enriched for genes involved in lignin biosynthesis. In all, 23 hub genes were identified based on a weighted gene coexpression network analysis of the 6,831 DEGs and their expression profiles at different time points after V. dahliae infection. Knockdown of Gh4CL30, one of the hub genes related to the lignin biosynthesis pathway, by virus-induced gene silencing, led to a decreased content of flavonoids, lignin, and S monomer but an increased content of G monomer, G/S lignin monomer, caffeic acid, and ferulic acid, and enhanced cotton resistance to V. dahliae. These results suggest that Gh4CL30 is a key gene modulating the outputs of different branches of the lignin biosynthesis pathway, and provide new insights into cotton resistance to V. dahliae.[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.

Local Rather than Global H3K27me3 Dynamics Are Associated with Differential Gene Expression in Verticillium dahliae.

Differential growth conditions typically trigger global transcriptional responses in filamentous fungi. Such fungal responses to environmental cues involve epigenetic regulation, including chemical histone modifications. It has been proposed that conditionally expressed genes, such as those that encode secondary metabolites but also effectors in pathogenic species, are often associated with a specific histone modification, lysine27 methylation of H3 (H3K27me3). However, thus far, no analyses on the global H3K27me3 profiles have been reported under differential growth conditions in order to assess if H3K27me3 dynamics govern differential transcription. Using chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing data from the plant-pathogenic fungus Verticillium dahliae grown in three in vitro cultivation media, we now show that a substantial number of the identified H3K27me3 domains globally display stable profiles among these growth conditions. However, we observe local quantitative differences in H3K27me3 ChIP-seq signals that are associated with a subset of differentially transcribed genes between media. Comparing the in vitro results to expression during plant infection suggests that in planta-induced genes may require chromatin remodeling to achieve expression. Overall, our results demonstrate that some loci display H3K27me3 dynamics associated with concomitant transcriptional variation, but many differentially expressed genes are associated with stable H3K27me3 domains. Thus, we conclude that while H3K27me3 is required for transcriptional repression, it does not appear that transcriptional activation requires the global erasure of H3K27me3. We propose that the H3K27me3 domains that do not undergo dynamic methylation may contribute to transcription through other mechanisms or may serve additional genomic regulatory functions. IMPORTANCE In many organisms, including filamentous fungi, epigenetic mechanisms that involve chemical and physical modifications of DNA without changing the genetic sequence have been implicated in transcriptional responses upon developmental or environmental cues. In fungi, facultative heterochromatin that can decondense to allow transcription in response to developmental changes or environmental stimuli is characterized by the trimethylation of lysine 27 on histone H3 (H3K27me3), and H3K27me3 has been implicated in transcriptional regulation, although the precise mechanisms and functions remain enigmatic. Based on ChIP and RNA sequencing data, we show for the soilborne broad-host-range vascular wilt plant-pathogenic fungus Verticillium dahliae that although some loci display H3K27me3 dynamics that can contribute to transcriptional variation, other loci do not show such a dependence. Thus, although we recognize that H3K27me3 is required for transcriptional repression, we also conclude that this mark is not a conditionally responsive global regulator of differential transcription upon responses to environmental cues.

Verticillium dahliae VdBre1 is required for cotton infection by modulating lipid metabolism and secondary metabolites.

The soil-borne ascomycete Verticillium dahliae causes wilt disease in more than two hundred dicotyledonous plants including the economically important crop cotton, and results in a severe reduction in cotton fiber yield and quality. During infection, V. dahliae secretes numerous secondary metabolites, which act as toxic factors to promote the infection process. However, the mechanism underlying how V. dahliae secondary metabolites regulate cotton infection remains largely unexplored. In this study, we report that VdBre1, an ubiquitin ligase (E3) enzyme to modify H2B, regulates radial growth and conidia production of V. dahliae. The VdBre1 deletion strains show nonpathogenic symptoms on cotton, and microscopic inspection and penetration assay indicated that penetration ability of the ∆VdBre1 strain was dramatically reduced. RNA-seq revealed that a total of 1643 differentially expressed genes between the ∆VdBre1 strain and the wild type strain V592, among which genes related to lipid metabolism were significantly overrepresented. Remarkably, the volume of lipid droplets in the ∆VdBre1 conidia was shown to be smaller than that of wild-type strains. Further metabolomics analysis revealed that the pathways of lipid metabolism and secondary metabolites, such as steroid biosynthesis and metabolism of terpenoids and polyketides, have dramatically changed in the ∆VdBre1 metabolome. Taken together, these results indicate that VdBre1 plays crucial roles in cotton infection and pathogenecity, by globally regulating lipid metabolism and secondary metabolism of V. dahliae.

Phytohormone crosstalk in the host-Verticillium interaction.

Functional genomics can be applied to shed light on the Brassica napus - Verticillium interaction. RNAseq data indicated already that abscisic acid (ABA) is apparently involved in the early oilseed rape response to fungal infection with Verticillium longisporum isolate 43 (Vl43). A calreticulin (CRT1a) was identified as novel susceptibility factor for Vl43 infecting both Arabidopsis and oilseed rape. CRT1a is involved in calcium homeostasis and contributes in the endoplasmatic reticulum to the unfolded protein response. The latter function could either affect the correct folding of other susceptibility factors or of negative regulators in ethylene (ET) signaling. Which CRT1a function is affected in the mutants is currently unknown, but both hypotheses can explain that crt1a loss-of-function mutants display increased resistance to V. longisporum and enhanced expression of ethylene signaling related genes. This indicates that besides other phytohormones such as ABA or salicylic acid (SA) also ET plays a critical role in the plant-Verticillium interaction, which might be exploited to improve plant resistance.

Verticillium dahliae VdTHI20, Involved in Pyrimidine Biosynthesis, Is Required for DNA Repair Functions and Pathogenicity.

Verticillium dahliae (V. dahliae) infects roots and colonizes the vascular vessels of host plants, significantly reducing the economic yield of cotton and other crops. In this study, the protein VdTHI20, which is involved in the thiamine biosynthesis pathway, was characterized by knocking out the corresponding VdTHI20 gene in V. dahliae via Agrobacterium tumefaciens-mediated transformation (ATMT). The deletion of VdTHI20 resulted in several phenotypic defects in vegetative growth and conidiation and in impaired virulence in tobacco seedlings. We show that VdTHI20 increases the tolerance of V. dahliae to UV damage. The impaired vegetative growth of ΔVdTHI20 mutant strains was restored by complementation with a functional copy of the VdTHI20 gene or by supplementation with additional thiamine. Furthermore, the root infection and colonization of the ΔVdTHI20 mutant strains were suppressed, as indicated by green fluorescent protein (GFP)-labelling under microscope observation. When the RNAi constructs of VdTHI20 were used to transform Nicotiana benthamiana, the transgenic lines expressing dsVdTHI20 showed elevated resistance to V. dahliae. Together, these results suggest that VdTHI20 plays a significant role in the pathogenicity of V. dahliae. In addition, the pathogenesis-related gene VdTHI20 exhibits potential for controlling V. dahliae in important crops.