Showing papers by "Steven J. Klosterman published in 2021"

A Single-Nucleotide Mutation in a GLUTAMATE RECEPTOR-LIKE Gene Confers Resistance to Fusarium Wilt in Gossypium hirsutum

Abstract: Fusarium wilt (FW) disease of cotton, caused by the fungus Fusarium oxysporum f. sp. vasinfectum (Fov), causes severe losses in cotton production worldwide. Though significant advancements have been made in development of FW-resistant Upland cotton (Gossypium hirsutum) in resistance screening programs, the precise resistance genes and the corresponding molecular mechanisms for resistance to Fov remain unclear. Herein it is reported that Fov7, a gene unlike canonical plant disease-resistance (R) genes, putatively encoding a GLUTAMATE RECEPTOR-LIKE (GLR) protein, confers resistance to Fov race 7 in Upland cotton. A single nucleotide polymorphism (SNP) (C/A) in GhGLR4.8, resulting in an amino acid change (L/I), is associated with Fov resistance. A PCR-based DNA marker (GhGLR4.8SNP(A/C) ) is developed and shown to cosegregate with the Fov resistance. CRISPR/Cas9-mediated knockout of Fov7 results in cotton lines extremely susceptible to Fov race 7 with a loss of the ability to induce calcium influx in response to total secreted proteins (SEPs) of Fov. Furthermore, coinfiltration of SEPs with GhGLR4.8A results in a hypersensitive response. This first report of a GLR-encoding gene that functions as an R gene provides a new insight into plant-pathogen interactions and a new handle to develop cotton cultivars with resistance to Fov race 7.

GhMYB4 downregulates lignin biosynthesis and enhances cotton resistance to Verticillium dahliae.

Abstract: GhMYB4 acts as a negative regulator in lignin biosynthesis, which results in alteration of cell wall integrity and activation of cotton defense response. Verticillium wilt of cotton (Gossypium hirsutum) caused by the soil-borne fungus Verticillium dahliae (V. dahliae) represents one of the most important constraints of cotton production worldwide. Mining of the genes involved in disease resistance and illuminating the molecular mechanisms that underlie this resistance is of great importance in cotton breeding programs. Defense-induced lignification in plants is necessary for innate immunity, and there are reports of a correlation between increased lignification and disease resistance. In this study, we present an example in cotton whereby plants with reduced lignin content also exhibit enhanced disease resistance. We identified a negative regulator of lignin synthesis, in cotton encoded in GhMYB4. Overexpression of GhMYB4 in cotton and Arabidopsis enhanced resistance to V. dahliae with reduced lignin deposition. Moreover, GhMYB4 could bind the promoters of several genes involved in lignin synthesis, such as GhC4H-1, GhC4H-2, Gh4CL-4, and GhCAD-3, and impair their expression. The reduction of lignin content in GhMYB4-overexpressing cotton led to alterations of cell wall integrity (CWI) and released more oligogalacturonides (OGs) which may act as damage-associated molecular patterns (DAMPs) to stimulate plant defense responses. In support of this hypothesis, exogenous application with polygalacturonic acid (PGA) in cotton activated biosynthesis of jasmonic acid (JA) and JA-mediated defense against V. dahliae, similar to that described for cotton plants overexpressing GhMYB4. This study provides a new candidate gene for cotton disease-resistant breeding and an increased understanding of the relationship between lignin synthesis, OG release, and plant immunity.

Cytotoxic function of xylanase VdXyn4 in the plant vascular wilt pathogen Verticillium dahliae.

Abstract: Phytopathogen xylanases play critical roles in pathogenesis, likely due to their ability to degrade plant structural barriers and manipulate host immunity. As an invader of plant xylem vessels, the fungus Verticillium dahliae is thought to deploy complex cell wall degrading enzymes. Comparative genomics analyses revealed that the V. dahliae genome encodes a family of six xylanases, each possessing a glycosyl hydrolase 11 domain, but the functions of these enzymes are undetermined. Characterizing gene deletion mutants revealed that only V. dahliae xylanase 4 (VdXyn4) degraded the plant cell wall and contributed to the virulence of V. dahliae. VdXyn4 displayed cytotoxic activity and induced a necrosis phenotype during the late stages of infection, leading to vein and petiole collapse that depended on the enzyme simultaneously localizing to nuclei and chloroplasts. The internalization of VdXyn4 was in conjunction with that of the plasma membrane complexLeucine-rich repeat (LRR)-receptor-like kinase suppressor of BIR1-1 (SOBIR1)/LRR-RLK BRI1-associated kinase-1 (BAK1), but we could not rule out the possibility that VdXyn4 may also act as an apoplastic effector. Immune signaling (in the SA-JA pathways) induced by VdXyn4 relative to that induced by known immunity effectors was substantially delayed. While cytotoxic activity could be partially suppressed by known effectors, they failed to impede necrosis in Nicotiana benthamiana. Thus, unlike typical effectors, cytotoxicity of VdXyn4 plays a crucial intracellular role at the late stages of V. dahliae infection and colonization, especially following pathogen entry into the xylem; this cytotoxic activity is likely conserved in the corresponding enzyme families in plant vascular pathogens.

Verticillium klebahnii and V. isaacii Isolates Exhibit Host-Dependent Biological Control of Verticillium Wilt Caused by V. dahliae

Abstract: Verticillium dahliae, the soilborne fungal pathogen, causes vascular wilt on many economically important crops, resulting in significant yield losses. V. klebahnii (isolate PD659) and V. isaacii (i...

Ancestral chromosomes for the Peronosporaceae inferred from a telomere-to-telomere genome assembly of Peronospora effusa

Abstract: We report the first telomere-to-telomere genome assembly for an oomycete. This assembly has extensive synteny with less complete genome assemblies of other oomycetes and will therefore serve as a reference genome for this taxon. Downy mildew disease of spinach, caused by the oomycete Peronospora effusa, causes major losses to spinach production. The 17 chromosomes of P. effusa were assembled telomere-to-telomere using Pacific Biosciences High Fidelity reads. Sixteen chromosomes are complete and gapless; Chromosome 15 contains one gap bridging the nucleolus organizer region. Putative centromeres were identified on all chromosomes. This new assembly enables a re-evaluation of the genomic composition of Peronospora spp.; the assembly was almost double the size and contained more repeat sequences than previously reported for any Peronospora spp. Genome fragments consistently under-represented in six previously reported assemblies of P. effusa typically encoded repeats. Some genes annotated as encoding effectors were organized into multigene clusters on several chromosomes. At least two effector-encoding genes were annotated on every chromosome. The intergenic distances between annotated genes were consistent with the two-speed genome hypothesis, with some effectors located in gene-sparse regions. The near-gapless assembly revealed apparent horizontal gene transfer from Ascomycete fungi. Gene order was highly conserved between P. effusa and the genetically oriented assembly of the oomycete Bremia lactucae. High levels of synteny were also detected with Phytophthora sojae. Many oomycete species may have similar chromosome organization; therefore, this genome assembly provides the foundation for genomic analyses of diverse oomycetes.

Transcriptome Variations in Verticillium dahliae in Response to Two Different Inorganic Nitrogen Sources.

Abstract: The fungus Verticillium dahliae causes vascular wilt disease on hundreds of plant species. The main focus of the research to control this fungus has been aimed at infection processes such as penetration peg formation and effector secretion, but the ability of the fungus to acquire and utilize nutrients are often overlooked and may hold additional potential to formulate new disease control approaches. Little is known about the molecular mechanisms of nitrogen acquisition and assimilation processes in V. dahliae. In this present study, RNA sequencing and gene expression analysis were used to examine differentially expressed genes in response to the different nitrogen sources, nitrate and ammonium, in V. dahliae. A total of 3244 and 2528 differentially expressed genes were identified in response to nitrate and ammonium treatments, respectively. The data indicated nitrate metabolism requires additional energy input while ammonium metabolism is accompanied by reductions in particular cellular processes. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses of DEGs during nitrate metabolism revealed that many of the genes encoded those involved in protein biosynthetic and metabolic processes, especially ribosome and RNA polymerase biosynthesis, but also other processes including transport and organonitrogen compound metabolism. Analysis of DEGs in the ammonium treatment indicated that cell cycle, oxidoreductase, and certain metabolic activities were reduced. In addition, DEGs participating in the utilization of both nitrate and ammonium were related to L-serine biosynthesis, energy-dependent multidrug efflux pump activity, and glycerol transport. We further showed that the mutants of three differentially expressed transcription factors (VdMcm1, VdHapX, and VDAG_08640) exhibited abnormal phenotypes under nitrate and ammonium treatment compared with the wild type strain. Deletion of VdMcm1 displayed slower growth when utilizing both nitrogen sources, while deletion of VdHapX and VDAG_08640 only affected nitrate metabolism, inferring that nitrogen assimilation required regulation of bZIP transcription factor family and participation of cell cycle. Taken together, our findings illustrate the convergent and distinctive regulatory mechanisms between preferred (ammonium) and alternative nitrogen (nitrate) metabolism at the transcriptome level, leading to better understanding of inorganic nitrogen metabolism in V. dahliae.

Dynamics of Verticillium dahliae race 1 population under managed agricultural ecosystems.

Abstract: Background Plant pathogens and their hosts undergo adaptive changes in managed agricultural ecosystems, by overcoming host resistance, but the underlying genetic adaptations are difficult to determine in natural settings. Verticillium dahliae is a fungal pathogen that causes Verticillium wilt on many economically important crops including lettuce. We assessed the dynamics of changes in the V. dahliae genome under selection in a long-term field experiment. Results In this study, a field was fumigated before the Verticillium dahliae race 1 strain (VdLs.16) was introduced. A derivative 145-strain population was collected over a 6-year period from this field in which a seggregating population of lettuce derived from Vr1/vr1 parents were evaluated. We de novo sequenced the parental genome of VdLs.16 strain and resequenced the derivative strains to analyze the genetic variations that accumulate over time in the field cropped with lettuce. Population genomics analyses identified 2769 single-nucleotide polymorphisms (SNPs) and 750 insertion/deletions (In-Dels) in the 145 isolates compared with the parental genome. Sequence divergence was identified in the coding sequence regions of 378 genes and in the putative promoter regions of 604 genes. Five-hundred and nine SNPs/In-Dels were identified as fixed. The SNPs and In-Dels were significantly enriched in the transposon-rich, gene-sparse regions, and in those genes with functional roles in signaling and transcriptional regulation. Conclusions Under the managed ecosystem continuously cropped to lettuce, the local adaptation of V. dahliae evolves at a whole genome scale to accumulate SNPs/In-Dels nonrandomly in hypervariable regions that encode components of signal transduction and transcriptional regulation.

Composition of the microbiomes from spinach seeds infested or non-infested with Peronospora effusa or Verticillium dahliae

Abstract: The worldwide distribution of plant seeds can disseminate beneficial and plant pathogenic microorganisms. This phenomenon is of particular concern where seed production is geographically isolated f...