Wenxian Sun

Within-Species Flagellin Polymorphism in Xanthomonas campestris pv campestris and Its Impact on Elicitation of Arabidopsis FLAGELLIN SENSING2–Dependent Defenses

Bacterial flagellins have been portrayed as a relatively invariant pathogen-associated molecular pattern. We have found within-species, within-pathovar variation for defense-eliciting activity of flagellins among Xanthomonas campestris pv campestris (Xcc) strains. Arabidopsis thaliana FLAGELLIN SENSING2 (FLS2), a transmembrane leucine-rich repeat kinase, confers flagellin responsiveness. The flg22 region was the only Xcc flagellin region responsible for detectable elicitation of Arabidopsis defense responses. A Val-43/Asp polymorphism determined the eliciting/noneliciting nature of Xcc flagellins (structural gene fliC). Arabidopsis detected flagellins carrying Asp-43 or Asn-43 but not Val-43 or Ala-43, and it responded minimally for Glu-43. Wild-type Xcc strains carrying nonrecognized flagellin were more virulent than those carrying a recognized flagellin when infiltrated into Arabidopsis leaf mesophyll, but this correlation was misleading. Isogenic Xcc fliC gene replacement strains expressing eliciting or noneliciting flagellins grew similarly, both in leaf mesophyll and in hydathode/vascular colonization assays. The plant FLS2 genotype also had no detectable effect on disease outcome when previously untreated plants were infected by Xcc. However, resistance against Xcc was enhanced if FLS2-dependent responses were elicited 1 d before Xcc infection. Prior immunization was not required for FLS2-dependent restriction of Pseudomonas syringae pv tomato. We conclude that plant immune systems do not uniformly detect all flagellins of a particular pathogen species and that Xcc can evade Arabidopsis FLS2-mediated defenses unless the FLS2 system has been activated by previous infections.

Identification and Mutational Analysis of Arabidopsis FLS2 Leucine-Rich Repeat Domain Residues That Contribute to Flagellin Perception

Mutational, phylogenetic, and structural modeling approaches were combined to develop a general method to study leucine-rich repeat (LRR) domains and were used to identify residues within the Arabidopsis thaliana FLAGELLIN-SENSING2 (FLS2) LRR that contribute to flagellin perception. FLS2 is a transmembrane receptor kinase that binds bacterial flagellin or a flagellin-based flg22 peptide through a presumed physical interaction within the FLS2 extracellular domain. Double-Ala scanning mutagenesis of solvent-exposed β-strand/β-turn residues across the FLS2 LRR domain identified LRRs 9 to 15 as contributors to flagellin responsiveness. FLS2 LRR-encoding domains from 15 Arabidopsis ecotypes and 20 diverse Brassicaceae accessions were isolated and sequenced. FLS2 is highly conserved across most Arabidopsis ecotypes, whereas more diversified functional FLS2 homologs were found in many but not all Brassicaceae accessions. flg22 responsiveness was correlated with conserved LRR regions using Conserved Functional Group software to analyze structural models of the LRR for diverse FLS2 proteins. This identified conserved spatial clusters of residues across the β-strand/β-turn residues of LRRs 12 to 14, the same area identified by the Ala scan, as well as other conserved sites. Site-directed randomizing mutagenesis of solvent-exposed β-strand/β-turn residues across LRRs 9 to 15 identified mutations that disrupt flg22 binding and showed that flagellin perception is dependent on a limited number of tightly constrained residues of LRRs 9 to 15 that make quantitative contributions to the overall phenotypic response.

Probing the Arabidopsis flagellin receptor : FLS2-FLS2 association and the contributions of specific domains to signaling function

Transmembrane LRR-RLKs are a major class of plant proteins. This study investigates the functional contributions of multiple FLS2 protein domains and modifications to provide insight into structure-function relationships of LRR-RLK proteins in general. FLAGELLIN SENSING2 (FLS2) is a transmembrane receptor kinase that activates antimicrobial defense responses upon binding of bacterial flagellin or the flagellin-derived peptide flg22. We find that some Arabidopsis thaliana FLS2 is present in FLS2-FLS2 complexes before and after plant exposure to flg22. flg22 binding capability is not required for FLS2-FLS2 association. Cys pairs flank the extracellular leucine rich repeat (LRR) domain in FLS2 and many other LRR receptors, and we find that the Cys pair N-terminal to the FLS2 LRR is required for normal processing, stability, and function, possibly due to undescribed endoplasmic reticulum quality control mechanisms. By contrast, disruption of the membrane-proximal Cys pair does not block FLS2 function, instead increasing responsiveness to flg22, as indicated by a stronger oxidative burst. There was no evidence for intermolecular FLS2-FLS2 disulfide bridges. Truncated FLS2 containing only the intracellular domain associates with full-length FLS2 and exerts a dominant-negative effect on wild-type FLS2 function that is dependent on expression level but independent of the protein kinase capacity of the truncated protein. FLS2 is insensitive to disruption of multiple N-glycosylation sites, in contrast with the related receptor EF-Tu RECEPTOR that can be rendered nonfunctional by disruption of single glycosylation sites. These and additional findings more precisely define the molecular mechanisms of FLS2 receptor function.

Specific adaptation of Ustilaginoidea virens in occupying host florets revealed by comparative and functional genomics

Ustilaginoidea virens (Cooke) Takah is an ascomycetous fungus that causes rice false smut, a devastating emerging disease worldwide. Here we report a 39.4 Mb draft genome sequence of U. virens that encodes 8,426 predicted genes. The genome has ~25% repetitive sequences that have been affected by repeat-induced point mutations. Evolutionarily, U. virens is close to the entomopathogenic Metarhizium spp., suggesting potential host jumping across kingdoms. U. virens possesses reduced gene inventories for polysaccharide degradation, nutrient uptake and secondary metabolism, which may result from adaptations to the specific floret infection and biotrophic lifestyles. Consistent with their potential roles in pathogenicity, genes for secreted proteins and secondary metabolism and the pathogen-host interaction database genes are highly enriched in the transcriptome during early infection. We further show that 18 candidate effectors can suppress plant hypersensitive responses. Together, our analyses offer new insights into molecular mechanisms of evolution, biotrophy and pathogenesis of U. virens.

Genetic diversity and population structure of rice pathogen Ustilaginoidea virens in China.

Rice false smut caused by the fungal pathogen Ustilaginoidea virens is becoming a destructive disease throughout major rice-growing countries. Information about its genetic diversity and population structure is essential for rice breeding and efficient control of the disease. This study compared the genome sequences of two U . virens isolates. Three SNP-rich genomic regions were identified as molecular markers that could be used to analyze the genetic diversity and population structure of U . virens in China. A total of 56 multilocus sequence types (haplotypes) were identified out of 162 representative isolates from 15 provinces covering five major rice-growing areas in China. However, the phylogeny, based on sequences at individual SNP-rich regions, strongly conflicted with each other and there were significant genetic differences between different geographical populations. Gene flow between the different geographical populations and genetic differentiation within each geographical population were also detected. In addition, genetic recombination and genetic isolation resulting from geographic separation was also found.

The HD-GYP Domain Protein RpfG of Xanthomonas oryzae pv. oryzicola Regulates Synthesis of Extracellular Polysaccharides that Contribute to Biofilm Formation and Virulence on Rice

Bacterial leaf streak caused by Xanthomonas oryzae pv. oryzicola (Xoc) is one of the most important diseases in rice. However, little is known about the pathogenicity mechanisms of Xoc. Here we have investigated the function of three HD-GYP domain regulatory proteins in biofilm formation, the synthesis of virulence factors and virulence of Xoc. Deletion of rpfG resulted in altered production of extracellular polysaccharides (EPS), abolished virulence on rice and enhanced biofilm formation, but had little effect on the secretion of proteases and motility. In contrast, mutational analysis showed that the other two HD-GYP domain proteins had no effect on virulence factor synthesis and tested phenotypes. Mutation of rpfG led to up-regulation of the type III secretion system and altered expression of three putative glycosyltransferase genes gumD, pgaC and xagB, which are part of operons directing the synthesis of different extracellular polysaccharides. The pgaABCD and xagABCD operons were greatly up-regulated in the Xoc ΔrpfG mutant, whereas the expression of the gum genes was unaltered or slightly enhanced. The elevated biofilm formation of the Xoc ΔrpfG mutant was dramatically reduced upon deletion of gumD, xagA and xagB, but not when pgaA and pgaC were deleted. Interestingly, only the ΔgumD mutant, among these single gene mutants, exhibits multiple phenotype alterations including reduced biofilm and EPS production and attenuated virulence on rice. These data indicate that RpfG is a global regulator that controls biofilm formation, EPS production and bacterial virulence in Xoc and that both gumD- and xagB-dependent EPS contribute to biofilm formation under different conditions.

Determination and Analysis of Ustiloxins A and B by LC-ESI-MS and HPLC in False Smut Balls of Rice

Ustiloxins are cyclopeptide mycotoxins produced by the pathogenic fungus Villosiclava virens of rice false smut. Ustiloxins A and B as two main mycotoxins were determined conveniently by LC-ESI-MS in the water extract from rice false smut balls which were mostly composed of the chlamydospores and mycelia of the pathogen. Both ustiloxins A and B in the water extract were also quantitatively analyzed by HPLC. This is the first report on the determination and analysis of ustiloxins A and B simultaneously by LC-ESI-MS and HPLC in false smut balls of rice.

Enhancement of innate immune system in monocot rice by transferring the dicotyledonous elongation factor Tu receptor EFR

The elongation factor Tu (EF-Tu) receptor (EFR) in cruciferous plants specifically recognizes the N-terminal acetylated elf18 region of bacterial EF-Tu and thereby activates plant immunity. It has been demonstrated that Arabidopsis EFR confers broad-spectrum bacterial resistance in the EFR transgenic solanaceous plants. Here, the transgenic rice plants (Oryza sativa L. ssp. japonica cv. Zhonghua 17) and cell cultures with constitutive expression of AtEFR were developed to investigate whether AtEFR senses EF-Tu and thus enhances bacterial resistance in the monocot plants. We demonstrated that the Xanthomonas oryzae-derived elf18 peptide induced oxidative burst and mitogen-activated protein kinase activation in the AtEFR transgenic rice cells and plants, respectively. Pathogenesis-related genes, such as OsPBZ1, were upregulated dramatically in transgenic rice plant and cell lines in response to elf18 stimulation. Importantly, pretreatment with elf18 triggered strong resistance to X. oryzae pv. oryzae in the transgenic plants, which was largely dependent on the AtEFR expression level. These plants also exhibited enhanced resistance to rice bacterial brown stripe, but not to rice fungal blast. Collectively, the results indicate that the rice plants with heterologous expression of AtEFR recognize bacterial EF-Tu and exhibit enhanced broad-spectrum bacterial disease resistance and that pattern recognition receptor-mediated immunity may be manipulated across the two plant classes, dicots and monocots.

Infection of Ustilaginoidea virens intercepts rice seed formation but activates grain-filling-related genes.

Abstract Rice false smut has become an increasingly serious disease in rice (Oryza sativa L.) production worldwide. The typical feature of this disease is that the fungal pathogen Ustilaginoidea virens (Uv) specifically infects rice flower and forms false smut ball, the ustiloxin‐containing ball‐like fungal colony, of which the size is usually several times larger than that of a mature rice seed. However, the underlying mechanisms of Uv‐rice interaction are poorly understood. Here, we applied time‐course microscopic and transcriptional approaches to investigate rice responses to Uv infection. The results demonstrated that the flower‐opening process and expression of associated transcription factors, including ARF6 and ARF8, were inhibited in Uv‐infected spikelets. The ovaries in infected spikelets were interrupted in fertilization and thus were unable to set seeds. However, a number of grain‐filling‐related genes, including seed storage protein genes, starch anabolism genes and endosperm‐specific transcription factors (RISBZ1 and RPBF), were highly transcribed as if the ovaries were fertilized. In addition, critical defense‐related genes like NPR1 and PR1 were downregulated by Uv infection. Our data imply that Uv may hijack host nutrient reservoir by activation of the grain‐filling network because of growth and formation of false smut balls.

Purification of Ustiloxins A and B from Rice False Smut Balls by Macroporous Resins

Ustiloxins are cyclopeptide mycotoxins produced by Villosiclava virens, the pathogenic fungus of rice false smut disease. Both resins SP207 and SP700 were screened to show the best adsorption and desorption properties for ustiloxins A and B among 20 commercial macroporous resins. Dynamic adsorption and desorption tests were carried out to optimize the process parameters. The optimal conditions for adsorption of resin SP207 were a processing volume as 32 bed volumes (BV), pH value of 4, and flow rate of 2 BV/h; and those for desorption of resin SP207 were a 40:60 (v/v) ratio of ethanol to water, an eluent volume of 4 BV, pH value of 4 and a flow rate of 3 BV/h. The optimal conditions for adsorption of resin SP700 were a processing volume of 26 BV, pH value as 4, flow rate of 2 BV/h; and those for desorption of resin SP700 were a 30:70 (v/v) ratio of ethanol to water solution as eluent, volume of 4 BV, pH value as 4 and flow rate of 2 BV/h. Under the optimal conditions; the purities of ustiloxins A and B obtained with resin SP207 increased 23.06-fold and 19.78-fold, respectively; and their recoveries were 96.67% and 81.25%; respectively. Similarly; the purities of ustiloxins A and B obtained with resin SP700 increased 14.75-fold and 15.33-fold and their recoveries were 93.65% and 88.64%; respectively. The results show that adsorption and desorption on SP207 and SP700 resins are effective strategies for purifying ustiloxins A and B. The developed methods are beneficial for large-scale preparation and purification of ustiloxins A and B from rice false smut balls.