The Genome of the Fungal Pathogen Verticillium dahliae Reveals Extensive Bacterial to Fungal Gene Transfer.
ABSTRACT: Horizontal gene transfer (HGT) involves the transmission of genetic material between distinct evolutionary lineages and can be an important source of biological innovation. Reports of interkingdom HGT to eukaryotic microbial pathogens have accumulated over recent years. Verticillium dahliae is a notorious plant pathogen that causes vascular wilt disease on hundreds of plant species, resulting in high economic losses every year. Previously, the effector gene Ave1 and a glucosyltransferase-encoding gene were identified as virulence factor-encoding genes that were proposed to be horizontally acquired from a plant and a bacterial donor, respectively. However, to what extent HGT contributed to the overall genome composition of V. dahliae remained elusive. Here, we systematically searched for evidence of interkingdom HGT events in the genome of V. dahliae and provide evidence for extensive horizontal gene acquisition from bacterial origin.
Project description:Wilt caused by Verticillium dahliae significantly reduces cotton yields, as host resistance in commercially cultivated Gossypium species is lacking. Understanding the molecular basis of disease resistance in non-commercial Gossypium species could galvanize the development of Verticillium wilt resistance in cultivated species. Nucleotide-binding site leucine-rich repeat (NBS-LRR) proteins play a central role in plant defence against pathogens. In this study, we focused on the relationship between a locus enriched with eight NBS-LRR genes and Verticillium wilt resistance in G. barbadense. Independent virus-induced gene silencing of each of the eight NBS-LRR genes in G. barbadense cultivar Hai 7124 revealed that silencing of GbaNA1 alone compromised the resistance of G. barbadense to V. dahliae isolate Vd991. In cultivar Hai 7124, GbaNA1 could be induced by V. dahliae isolate Vd991 and by ethylene, jasmonic acid and salicylic acid. Nuclear protein localization of GbaNA1 was demonstrated by transient expression. Sequencing of the GbaNA1 orthologue in nine G. hirsutum accessions revealed that all carried a non-functional allele, caused by a premature peptide truncation. In addition, all 10 G. barbadense and nine G. hirsutum accessions tested carried a full-length (?1140 amino acids) homologue of the V. dahliae race 1 resistance gene Gbve1, although some sequence polymorphisms were observed. Verticillium dahliae Vd991 is a non-race 1 isolate that lacks the Ave1 gene. Thus, the resistance imparted by GbaNA1 appears to be mediated by a mechanism distinct from recognition of the fungal effector Ave1.
Project description:Fungal plant pathogens secrete effector molecules to establish disease on their hosts, and plants in turn use immune receptors to try to intercept these effectors. The tomato immune receptor Ve1 governs resistance to race 1 strains of the soil-borne vascular wilt fungi Verticillium dahliae and Verticillium albo-atrum, but the corresponding Verticillium effector remained unknown thus far. By high-throughput population genome sequencing, a single 50-Kb sequence stretch was identified that only occurs in race 1 strains, and subsequent transcriptome sequencing of Verticillium-infected Nicotiana benthamiana plants revealed only a single highly expressed ORF in this region, designated Ave1 (for Avirulence on Ve1 tomato). Functional analyses confirmed that Ave1 activates Ve1-mediated resistance and demonstrated that Ave1 markedly contributes to fungal virulence, not only on tomato but also on Arabidopsis. Interestingly, Ave1 is homologous to a widespread family of plant natriuretic peptides. Besides plants, homologous proteins were only found in the bacterial plant pathogen Xanthomonas axonopodis and the plant pathogenic fungi Colletotrichum higginsianum, Cercospora beticola, and Fusarium oxysporum f. sp. lycopersici. The distribution of Ave1 homologs, coincident with the presence of Ave1 within a flexible genomic region, strongly suggests that Verticillium acquired Ave1 from plants through horizontal gene transfer. Remarkably, by transient expression we show that also the Ave1 homologs from F. oxysporum and C. beticola can activate Ve1-mediated resistance. In line with this observation, Ve1 was found to mediate resistance toward F. oxysporum in tomato, showing that this immune receptor is involved in resistance against multiple fungal pathogens.
Project description:Okra (Abelmoschus esculentus (L.) Moench) has gained more popularity as an economically significant plant for its nutritional and medicinal value, especially in China. During 2014-2016, the root disease of okra was discovered in four okra commercial fields surveyed in China. A fungul was isolated from the infected tissues, and was identified by Verticillium dahliae based on morphological characteristics. Pathogenicity test demonstrated that the fungus was pathogenic on okra, and fulfilled Koch's postulates. The analysis of three sequences revealed 99-100% identity with the reported V. dahliae strain in GenBank. Neighbor-joining analysis of the gene sequences revealed that the representative isolates were clustered with V. dahliae. To the best of our knowledge, this is the first report of Verticillium wilt of okra in China.
Project description:Plant-pathogenic microbes secrete effector molecules to establish themselves on their hosts, whereas plants use immune receptors to try and intercept such effectors in order to prevent pathogen colonization. The tomato cell surface-localized receptor Ve1 confers race-specific resistance against race 1 strains of the soil-borne vascular wilt fungus Verticillium dahliae which secrete the Ave1 effector. Here, we describe the cloning and characterization of Ve1 homologues from tobacco (Nicotiana glutinosa), potato (Solanum tuberosum), wild eggplant (Solanum torvum) and hop (Humulus lupulus), and demonstrate that particular Ve1 homologues govern resistance against V. dahliae race 1 strains through the recognition of the Ave1 effector. Phylogenetic analysis shows that Ve1 homologues are widely distributed in land plants. Thus, our study suggests an ancient origin of the Ve1 immune receptor in the plant kingdom.
Project description:The recognition of pathogen effectors by plant immune receptors leads to the activation of immune responses that often include a hypersensitive response (HR): rapid and localized host cell death surrounding the site of attempted pathogen ingress. We have demonstrated previously that the recognition of the Verticillium dahliae effector protein Ave1 by the tomato immune receptor Ve1 triggers an HR in tomato and tobacco. Furthermore, we have demonstrated that tomato Ve1 provides Verticillium resistance in Arabidopsis upon Ave1 recognition. In this study, we investigated whether the co-expression of Ve1 and Ave1 in Arabidopsis results in an HR, which could facilitate a forward genetics screen. Surprisingly, we found that the co-expression of Ve1 and Ave1 does not induce an HR in Arabidopsis. These results suggest that an HR may occur as a consequence of Ve1/Ave1-induced immune signalling in tomato and tobacco, but is not absolutely required for Verticillium resistance.
Project description:Verticillium wilt, caused by soil-borne fungi of the genus Verticillium, is an economically important disease that affects a wide range of host plants. Unfortunately, host resistance against Verticillium wilts is not available for many plant species, and the disease is notoriously difficult to combat. Host-induced gene silencing (HIGS) is an RNA interference (RNAi)-based process in which small RNAs are produced by the host plant to target parasite transcripts. HIGS has emerged as a promising strategy for the improvement of plant resistance against pathogens by silencing genes that are essential for these pathogens. Here, we assessed whether HIGS can be utilized to suppress Verticillium wilt disease by silencing three previously identified virulence genes of V. dahliae (encoding Ave1, Sge1 and NLP1) through the host plants tomato and Arabidopsis. In transient assays, tomato plants were agroinfiltrated with Tobacco rattle virus (TRV) constructs to target V. dahliae transcripts. Subsequent V. dahliae inoculation revealed the suppression of Verticillium wilt disease on treatment with only one of the three TRV constructs. Next, expression of RNAi constructs targeting transcripts of the same three V. dahliae virulence genes was pursued in stable transgenic Arabidopsis thaliana plants. In this host, V. dahliae inoculation revealed reduced Verticillium wilt disease in two of the three targets. Thus, our study suggests that, depending on the target gene chosen, HIGS against V. dahliae is operational in tomato and A. thaliana plants and may be exploited to engineer resistance in Verticillium wilt-susceptible crops.
Project description:Pathogenic Verticillium species are economically important plant pathogens that cause vascular wilt diseases in hundreds of plant species. The Ve1 gene of tomato confers resistance against race 1 strains of Verticillium dahliae and V. albo-atrum. Ve1 encodes an extracellular leucine-rich repeat (eLRR) receptor-like protein (RLP) that serves as a cell surface receptor for recognition of the recently identified secreted Verticillium effector Ave1. To investigate recognition of Ave1 by Ve1, alanine scanning was performed on the solvent exposed ?-strand/?-turn residues across the eLRR domain of Ve1. In addition, alanine scanning was also employed to functionally characterize motifs that putatively mediate protein-protein interactions and endocytosis in the transmembrane domain and the cytoplasmic tail of the Ve1 protein. Functionality of the mutant proteins was assessed by screening for the occurrence of a hypersensitive response upon co-expression with Ave1 upon Agrobacterium tumefaciens-mediated transient expression (agroinfiltration). In order to confirm the agroinfiltration results, constructs encoding Ve1 mutants were transformed into Arabidopsis and the transgenes were challenged with race 1 Verticillium. Our analyses identified several regions of the Ve1 protein that are required for functionality.
Project description:MiRNAs in animals and plants play crucial roles in diverse developmental processes under both normal and stress conditions. miRNA-like small RNAs (milRNAs) identified in some fungi remain functionally uncharacterized. Here, we identified a number of milRNAs in Verticillium dahliae, a soil-borne fungal pathogen responsible for devastating wilt diseases in many crops. Accumulation of a V. dahliae milRNA1, named VdmilR1, was detected by RNA gel blotting. We show that the precursor gene VdMILR1 is transcribed by RNA polymerase II and is able to produce the mature VdmilR1, in a process independent of V. dahliae DCL (Dicer-like) and AGO (Argonaute) proteins. We found that an RNaseIII domain-containing protein, VdR3, is essential for V. dahliae and participates in VdmilR1 biogenesis. VdmilR1 targets a hypothetical protein-coding gene, VdHy1, at the 3'UTR for transcriptional repression through increased histone H3K9 methylation of VdHy1. Pathogenicity analysis reveals that VdHy1 is essential for fungal virulence. Together with the time difference in the expression patterns of VdmilR1 and VdHy1 during fungal infection in cotton plants, our findings identify a novel milRNA, VdmilR1, in V. dahliae synthesized by a noncanonical pathway that plays a regulatory role in pathogenicity and uncover an epigenetic mechanism for VdmilR1 in regulating a virulence target gene. This article is part of the theme issue 'Biotic signalling sheds light on smart pest management'.
Project description:Long noncoding RNAs (lncRNAs) have several known functions in plant development, but their possible roles in responding to plant disease remain largely unresolved. In this study, we described a comprehensive disease-responding lncRNA profiles in defence against a cotton fungal disease Verticillium dahliae. We further revealed the conserved and specific characters of disease-responding process between two cotton species. Conservatively for two cotton species, we found the expression dominance of induced lncRNAs in the Dt subgenome, indicating a biased induction pattern in the co-existing subgenomes of allotetraploid cotton. Comparative analysis of lncRNA expression and their proposed functions in resistant Gossypium barbadense cv. '7124' versus susceptible Gossypium hirsutum cv. 'YZ1' revealed their distinct disease response mechanisms. Species-specific (LS) lncRNAs containing more SNPs displayed a fiercer inducing level postinfection than the species-conserved (core) lncRNAs. Gene Ontology enrichment of LS lncRNAs and core lncRNAs indicates distinct roles in the process of biotic stimulus. Further functional analysis showed that two core lncRNAs, GhlncNAT-ANX2- and GhlncNAT-RLP7-silenced seedlings, displayed an enhanced resistance towards V. dahliae and Botrytis cinerea, possibly associated with the increased expression of LOX1 and LOX2. This study represents the first characterization of lncRNAs involved in resistance to fungal disease and provides new clues to elucidate cotton disease response mechanism.
Project description:Small RNAs (sRNAs, including small interfering RNAs [siRNAs] and micro RNAs [miRNAs]) are key mediators of RNA silencing (or RNA interference), which play important roles in plant development and response to biotic and abiotic stimulation. Verticillium wilt is a plant vascular disease caused by the soil-borne fungal pathogens, such as Verticillium dahliae. We previously reported that V. dahliae infection increased two plant endogenous miRNAs that were exported to fungal cell to silence virulence genes. To investigate plant sRNAs in genome-wide response to V. dahliae infection, in this study, we constructed two sRNA libraries from Arabidopsis roots with and without V. dahliae infection, respectively. In total, 31 conserved miRNAs were found to be differentially expressed during the early stage of infection with V. dahliae using sRNA sequencing. Among these, the expression levels of miR160, miR164, miR166, miR167, miR390 and miR156h were confirmed by northern blot. Reverse transcription quantitative real time polymerase chain reaction results showed that the induction of miRNAs (miR160, miR164, miR166 and miR167) upon V. dahliae infection downregulated the expression of their targeted genes (ARF10, NAC1, PHV and ARF6), respectively. In addition, we identified specific phased siRNAs generated from distinct regions of two libraries. Profiling of these miRNAs and sRNAs lay the foundation for further understanding and utilising the host-induced gene silencing strategy to control plant vascular pathogens.