Tomonori Shiraishi

RRS1 and RPS4 provide a dual Resistance-gene system against fungal and bacterial pathogens

Colletotrichum higginsianum is a fungal pathogen that infects a wide variety of cruciferous plants, causing important crop losses. We have used map-based cloning and natural variation analysis of 19 Arabidopsis ecotypes to identify a dominant resistance locus against C. higginsianum. This locus named RCH2 (for recognition of C. higginsianum) maps in an extensive cluster of disease-resistance loci known as MRC-J in the Arabidopsis ecotype Ws-0. By analyzing natural variations within the MRC-J region, we found that alleles of RRS1 (resistance to Ralstonia solanacearum 1) from susceptible ecotypes contain single nucleotide polymorphisms that may affect the encoded protein. Consistent with this finding, two susceptible mutants, rrs1-1 and rrs1-2, were identified by screening a T-DNA-tagged mutant library for the loss of resistance to C. higginsianum. The screening identified an additional susceptible mutant (rps4-21) that has a 5-bp deletion in the neighboring gene, RPS4-Ws, which is a well-characterized R gene that provides resistance to Pseudomonas syringae pv. tomato strain DC3000 expressing avrRps4 (Pst-avrRps4). The rps4-21/rrs1-1 double mutant exhibited similar levels of susceptibility to C. higginsianum as the single mutants. We also found that both RRS1 and RPS4 are required for resistance to R. solanacearum and Pst-avrRps4. Thus, RPS4-Ws and RRS1-Ws function as a dual resistance gene system that prevents infection by three distinct pathogens.

Flagellin Glycosylation Island in Pseudomonas syringae pv. glycinea and Its Role in Host Specificity

The deduced amino acid sequences of the flagellins of Pseudomonas syringae pv. tabaci and P. syringae pv. glycinea are identical; however, their abilities to induce a hypersensitive reaction are clearly different. The reason for the difference seems to depend on the posttranslational modification of the flagellins. To investigate the role of this posttranslational modification in the interactions between plants and bacterial pathogens, we isolated genes that are potentially involved in the posttranslational modification of flagellin in P. syringae pv. glycinea (glycosylation island); then defective mutants with mutations in these genes were generated. There are three open reading frames in the glycosylation island, designated orf1, orf2, and orf3. orf1 and orf2 encode putative glycosyltransferases, and mutants with defects in these open reading frames, deltaorf1 and deltaorf2, secreted nonglycosylated and slightly glycosylated flagellins, respectively. Inoculation tests performed with these mutants and original nonhost tobacco leaves revealed that deltaorf1 and deltaorf2 could grow on tobacco leaves and caused symptom-like changes. In contrast, these mutants failed to cause symptoms on original host soybean leaves. These data indicate that putative glycosyltransferases encoded in the flagellin glycosylation island are strongly involved in recognition by plants and could be the specific determinants of compatibility between phytopathogenic bacteria and plant species.

RCH1, a Locus in Arabidopsis That Confers Resistance to the Hemibiotrophic Fungal Pathogen Colletotrichum higginsianum

When challenged with the crucifer pathogen Colletotrichum higginsianum, Arabidopsis thaliana ecotype Columbia (Col-0) was colonized by the fungus within 2 to 3 days, developing brown necrotic lesions surrounded by a yellow halo. Lesions spread from the inoculation site within 3 to 4 days, and subsequently continued to expand until they covered the entire leaf. Electron microscopy confirmed that C. higginsianum is a hemibiotroph on Arabidopsis, feeding initially on living cells as a biotroph before switching to a necrotrophic mode of growth. A collection of 37 ecotypes of Arabidopsis varied in their responses to infection by C. higginsianum. The ecotype Eil-0 was highly resistant, with symptoms limited to necrotic flecking and with only very limited fungal colonization. Analyses suggested that the hypersensitive response and reactive oxygen species may be important in this defense response. Expression analyses with cDNA microarrays indicated that the defense reaction depends primarily on the jasmonic acid- and ethylene-dependent signaling pathways and, to a lesser extent, on the salicylate-dependent pathway. Crosses between the Eil-0 and Col-0 ecotypes suggested that the resistance in Eil-0 was dominant and was conferred by a single locus, which we named RCH1. RCH1 is the first resistance locus to be identified from Arabidopsis against the hemibiotrophic fungus genus Colletotrichum.

cDNA cloning and characterization of tobacco ABC transporter: NtPDR1 is a novel elicitor-responsive gene1

We isolated an INF1 elicitin‐inducible cDNA encoding a pleiotropic drug resistance (PDR)‐type ATP‐binding cassette (ABC) transporter homolog (NtPDR1) in suspension‐cultured tobacco Bright Yellow‐2 (BY‐2) cells by application of differential display PCR. The NtPDR1 (Nicotiana tabacum PDR protein 1) gene also encodes a 162 kDa protein that includes two putative hydrophilic domains containing the ABC signature motif and two putative hydrophobic domains. Expression of the NtPDR1 gene was rapidly and strongly activated by treatment of BY‐2 cells with INF1 elicitin. Further, treatment of BY‐2 cells with flagellin, a bacterial proteinaceous hypersensitive reaction elicitor, or yeast extract, a general elicitor, also induced NtPDR1 gene expression. These results indicate that NtPDR1 may be involved in the general defense response in tobacco. This is the first report that microbial elicitors induce the expression of a plant ABC transporter gene.

Identification of glycosylation genes and glycosylated amino acids of flagellin in Pseudomonas syringae pv. tabaci

A glycosylation island is a genetic region required for glycosylation. The glycosylation island of flagellin in Pseudomonas syringae pv. tabaci 6605 consists of three orfs: orf1, orf2 and orf3. Orf1 and orf2 encode putative glycosyltransferases, and their deletion mutants, Δorf1 and Δorf2, exhibit deficient flagellin glycosylation or produce partially glycosylated flagellin respectively. Digestion of glycosylated flagellin from wild‐type bacteria and non‐glycosylated flagellin from Δorf1 mutant using aspartic N‐peptidase and subsequent HPLC analysis revealed candidate glycosylated amino acids. By generation of site‐directed Ser/Ala‐substituted mutants, all glycosylated amino acid residues were identified at positions 143, 164, 176, 183, 193 and 201. Matrix‐assisted laser desorption/ionization time of flight (MALDI‐TOF) mass spectrometry (MS) analysis revealed that each glycan was about 540 Da. While all glycosylation‐defective mutants retained swimming ability, swarming ability was reduced in the Δorf1, Δorf2 and Ser/Ala‐substituted mutants. All glycosylation mutants were also found to be impaired in the ability to adhere to a polystyrene surface and in the ability to cause disease in tobacco. Based on the predicted tertiary structure of flagellin, S176 and S183 are expected to be located on most external surface of the flagellum. Thus the effect of Ala‐substitution of these serines is stronger than that of other serines. These results suggest that glycosylation of flagellin in P. syringae pv. tabaci 6605 is required for bacterial virulence. It is also possible that glycosylation of flagellin may mask elicitor function of flagellin molecule.

Agrobacterium tumefaciens-mediated transformation for random insertional mutagenesis in Colletotrichum lagenarium

Random insertional mutagenesis using a marker DNA fragment is an effective method for identifying fungal genes relevant to morphogenesis, metabolism, and so on. Agrobacterium tumefaciens-mediated transformation (AtMT) has long been used as a tool for the genetic modification of a wide range of plant species. Recent study has indicated that A. tumefaciens could transfer T-DNA not only to plant cells but also to fungal cells. In this study, AtMT was applied to Colletotrichum lagenarium for random insertional mutagenesis. We constructed a binary vector pBIG2RHPH2 carrying a hygromycin-resistant gene cassette between the right and left borders of T-DNA. Optimal co-cultivation of C. lagenarium wild-type 104-T with pBIG2RHPH2-introduced A. tumefaciens C58C1 led to the production of 150–300 hygromycin-resistant transformants per 106 conidia. Southern blot analysis revealed that T-DNA was mainly integrated at a single site in the genome and at different sites in transformants. The T-DNA inserts showed small truncations of either end, but the hygromycin-resistant gene cassette inside the T-DNA was generally intact. The mode of T-DNA insertion described above resulted in highly efficient gene recovery from the transformants by thermal asymmetrical interlaced-polymerase chain reaction. The fungal genomic DNA segments flanking T-DNA were identified from five of eight mutants that had defective melanin biosynthesis. The sequence from one of the segments was identical to that of the melanin biosynthesis gene PKS1 of C. lagenarium, which we previously characterized. These results strongly support our notion that AtMT is a possible tool for tagging genes relevant to pathogenicity in the plant pathogenic fungus C. lagenarium.

The ΔfliD mutant of Pseudomonas syringae pv. tabaci, which secretes flagellin monomers, induces a strong hypersensitive reaction (HR) in non-host tomato cells

To investigate the role of flagella and monomer flagellin in the interaction between Pseudomonas syringae pv. tabaci and plants, non-polar fliC and fliD mutants were produced. The ORFs for fliC and fliD are deleted in the ΔfliC and ΔfliD mutants, respectively. Both mutants lost all flagella and were non-motile. The ΔfliC mutant did not produce flagellin, whereas the ΔfliD mutant, which lacks the HAP2 protein, secreted large amounts of monomer flagellin into the culture medium. Inoculation of non-host tomato leaves with wild-type P. syringae pv. tabaci or the ΔfliD mutant induced a hypersensitive reaction (HR), whereas the ΔfliC mutant propagated and caused characteristic symptom-like changes. In tomato cells in suspension culture, wild-type P. syringae pv. tabaci induced slight, visible HR-like changes. The ΔfliC mutant did not induce HR, but the ΔfliD mutant induced a remarkably strong HR. Expression of the hsr203J gene was rapidly and strongly induced by inoculation with the ΔfliD mutant, compared to inoculation with wild-type P. syringae pv. tabaci. Furthermore, introduction of the fliC gene into the ΔfliC mutant restored motility and HR-inducing ability in tomato. These results, together with our previous study, suggest that the flagellin monomer of pv. tabaci acts as a strong elicitor to induce HR-associated cell death in non-host tomato cells.

The Colletotrichum lagenarium Ste12-Like Gene CST1 Is Essential for Appressorium Penetration

Colletotrichum lagenarium is the causal agent of anthracnose of cucumber. This fungus produces a darkly melanized infection structure, appressoria, to penetrate the host leaves. The C. lagenarium CMK1 gene, a homologue of the Saccharomyces cerevisiae FUS3/KSS1 mitogen-activated protein (MAP) kinase genes, was shown to regulate conidial germination, appressorium formation, and invasive growth. In S. cerevisiae, Ste12p is known to be a transcriptional factor downstream of Fus3p/Kss1p MAP kinases. To evaluate the CMK1 MAP kinase pathway, we isolated the Ste12 homologue CST1 gene from C. lagenarium and characterized. The cst1delta strains were nonpathogenic on intact host leaves, but could form lesions when inoculated on wounded leaves. Conidia of the cst1delta strains could germinate and form melanized appressoria on both host leaf surface and artificial cellulose membrane, but could not produce infectious hyphae from appressoria, suggesting that CST1 is essential for appressorium penetration in C. lagenarium. In addition, matured appressoria of the cst1delta strains contained an extremely low level of lipid droplets compared with that of the wild-type strain. Lipid droplets were abundant in conidia of the cst1delta strains, but rapidly disappeared during appressorium formation. This misscheduled lipid degradation might be related to the failure of appressorium penetration in the cst1delta strain.

Superoxide generation in extracts from isolated plant cell walls is regulated by fungal signal molecules.

ABSTRACT Fractions solubilized with NaCl from cell walls of pea and cowpea plants catalyzed the formation of blue formazan from nitroblue tetrazolium. Because superoxide dismutase decreased formazan production by over 90%, superoxide anion (O(2) ) may participate in the formation of formazan in the solubilized cell wall fractions. The formazan formation in the fractions solubilized from pea and cowpea cell walls was markedly reduced by exclusion of NAD(P)H, manganese ion, or p-coumaric acid from the reaction mixture. The formazan formation was severely inhibited by salicylhydroxamic acid and catalase, but not by imidazole, pyridine, quinacrine, and diphenyleneiodonium. An elicitor preparation from the pea pathogen Mycosphaerella pinodes enhanced the activities of formazan formation nonspecifically in both pea and cowpea fractions. The suppressor preparation from M. pinodes inhibited the activity in the pea fraction in the presence or absence of the elicitor. In the cowpea fraction, however, the suppressor did not inhibit the elicitor-enhanced activity, and the suppressor alone stimulated formazan formation. These results indicated that O(2) generation in the fractions solubilized from pea and cowpea cell walls seems to be catalyzed by cell wall-bound peroxidase(s) and that the plant cell walls alone are able to respond to the elicitor non-specifically and to the suppressor in a species-specific manner, suggesting the plant cell walls may play an important role in determination of plant-fungal pathogen specificity.

Modulation of defense signal transduction by flagellin-induced WRKY41 transcription factor in Arabidopsis thaliana

Flagellin, a component of the flagellar filament of Pseudomonas syringae pv. tabaci 6605 (Pta), induces hypersensitive reaction in its non-host Arabidopsis thaliana. We identified the WRKY41 gene, which belongs to a multigene family encoding WRKY plant-specific transcription factors, as one of the flagellin-inducible genes in A. thaliana. Expression of WRKY41 is induced by inoculation with the incompatible pathogen P. syringae pv. tomato DC3000 (Pto) possessing AvrRpt2 and the non-host pathogens Pta within 6-h after inoculation, but not by inoculation with the compatible Pto. Expression of WRKY41 was also induced by inoculation of A. thaliana with an hrp-type three secretion system (T3SS)-defective mutant of Pto, indicating that effectors produced by T3SS in the Pto wild-type suppress the activation of WRKY41. Arabidopsis overexpressing WRKY41 showed enhanced resistance to the Pto wild-type but increased susceptibility to Erwinia carotovora EC1. WRKY41-overexpressing Arabidopsis constitutively expresses the PR5 gene, but suppresses the methyl jasmonate-induced PDF1.2 gene expression. These results demonstrate that WRKY41 may be a key regulator in the cross talk of salicylic acid and jasmonic acid pathways.