Chao-Ying Chen

Dimethyl disulfide is an induced systemic resistance elicitor produced by Bacillus cereus C1L.

BACKGROUND Bacillus cereus C1L is a plant growth-promoting rhizobacterium and can elicit induced systemic resistance (ISR) in plants against necrotrophic pathogens. However, little is known about ISR elicitors produced by B. cereus C1L, and no ISR elicitor has been identified and characterised. Therefore, the objective of this study is to identify volatile ISR elicitor(s) produced by B. cereus C1L. RESULTS The volatile metabolites produced by B. cereus C1L were extracted, separated and identified by solid-phase microextraction, gas chromatography and mass spectrometry. Dimethyl disulfide (DMDS) was the only separated metabolite being determined. Afterwards, application of DMDS by means of soil drench significantly protected tobacco and corn plants against Botrytis cinerea and Cochliobolus heterostrophus, respectively, under greenhouse conditions. The results reveal that DMDS could play an important role in ISR by B. cereus C1L. CONCLUSION This is the first report of DMDS as an elicitor produced by an ISR-eliciting B. cereus strain and its ability to suppress plant fungal diseases under greenhouse conditions. It is suggested that DMDS has potential for practical use in controlling plant foliar diseases besides soil fumigation.

Evidence of Induced Systemic Resistance Against Botrytis elliptica in Lily

Lily leaf blight, caused by Botrytis elliptica, is an important fungal disease in Taiwan. In order to identify an effective, nonfungicide method to decrease disease incidence in Lilium formosanum, the efficacy of rhizobacteria eliciting induced systemic resistance (ISR) was examined in this study. Over 300 rhizobacteria were isolated from the rhizosphere of L. formosanum healthy plants and 63 were identified by the analysis of fatty acid profiles. Disease suppressive ability of 13 strains was demonstrated by soil drench application of bacterial suspensions to the rhizosphere of L. formosanum seedlings. Biocontrol experiments were carried out with Bacillus cereus and Pseudomonas putida strains on L. formosanum and Lilium Oriental hybrid cvs. Acapulco and Star Gazer in greenhouse and field studies. Plants treated with B. cereus strain C1L showed that protection against B. elliptica on L. formosanum could last for at least 10 days and was consistent with high populations of B. cereus on lily roots. Analysis of the expression of LfGRP1 and LsGRP1, encoding glycine-rich protein associated with L. formosanum and cv. Star Gazer, respectively, revealed different responses induced by B. cereus or by the pathogen B. elliptica, suggesting that plant defense responses elicited by each follows a different signaling pathway. According to the results of biocontrol assays and LfGRP1/LsGRP1 gene expression analyses with culture filtrates of B. cereus strain C1L, we propose that eliciting factors of ISR are generated by B. cereus and some of them exhibit thermostable and heat-tolerant traits. This is the first report about ISR-eliciting rhizobacteria and factors effective for foliar disease suppression in lily.

Disease resistance to bacterial pathogens affected by the amount of ferredoxin-I protein in plants.

SUMMARY Ferredoxin-I (Fd-I) is a fundamental protein that is involved in several metabolic pathways. The amount of Fd-I found in plants is generally regulated by environmental stress, including biotic and abiotic events. In this study, the correlation between quantity of Fd-I and plant disease resistance was investigated. Fd-I levels were increased by inoculation with Pseudomonas syringae pv. syringae but were reduced by Erwinia carotovora ssp. carotovora. Transgenic tobacco over-expressing Fd-I with the sense sweet pepper Fd-I gene (pflp) was resistant to E. carotovora ssp. carotovora and the saprophytic bacterium P. fluorescens. By contrast, transgenic tobacco with reduced total Fd-I and the antisense pflp gene was susceptible to E. carotovora ssp. carotovora and P. fluorescens. Both of these transgenic tobaccos were resistant to P. syringae pv. syringae. By contrast, the mutated E. carotovora ssp. carotovora, with a defective harpin protein, was able to invade the sense-pflp transgenic tobacco as well as the non-transgenic tobacco. An in vitro kinase assay revealed that harpin could activate unidentified kinases to phosphorylate PFLP. These results demonstrate that Fd-I plays an important role in the disease defence mechanism.

The Arabidopsis short-chain dehydrogenase/reductase 3, an ABSCISIC ACID DEFICIENT 2 homolog, is involved in plant defense responses but not in ABA biosynthesis

ABSCISIC ACID DEFICIENT2 (ABA2) encodes a short-chain dehydrogenase/reductase1 (SDR1) that catalyzes the multi-step conversion of xanthoxin to abscisic aldehyde during abscisic acid (ABA) biosynthesis in Arabidopsis thaliana. In this study, AtSDR2 and AtSDR3, the two closest homologs to AtABA2, were investigated for their potential role in ABA biosynthesis. AtSDR2 showed undetectable transcription in plants grown under normal conditions or under stress. AtSDR3 and AtABA2 have different spatial and temporal expression patterns. Complementation testing demonstrated that the pABA2::SDR3 transgene failed to complement the aba2 mutant phenotype, and that transgenic plants showed the same levels of ABA as the aba2 mutants. These data suggest that AtSDR3 confers no functional redundancy to AtABA2 in ABA biosynthesis. Interestingly, microarray data derived from Genevestigator suggested that AtSDR3 might have a function that is related to plant defense. Pseudomonas syringae pv. tomato (Pst) DC3000 infection and systemic acquired resistance (SAR) activator application further demonstrated that AtSDR3 plays an important role in plant defense responses at least partially through the regulation of AtPR-1 gene expression.

Identification and transcriptional analysis of genes involved in Bacillus cereus-induced systemic resistance in Lilium

Bacillus cereus C1L was demonstrated to induce systemic resistance in Lilium formosanum against leaf blight caused by Botrytis elliptica. Suppression subtractive hybridization library of L. formosanum triggered by B. cereus C1L were screened and 3 differentially expressed genes were identified. Based on sequence analysis, these genes encoding putative glycine-rich protein, metallothionein-like protein, and PsbR protein of photosystem 2, were designated LfGRP1, LfMT1, and LfPsbR, respectively. The results of Northern blot analysis showed that expressions of LfGRP1, LfMT1 and LfPsbR increased in response to B. elliptica infection. On the other hand, expression of LfMT1 increased but expressions of LfGRP1 and LfPsbR decreased when the rhizosphere of L. formosanum was drenched with suspension of B. cereus C1L with or without subsequent challenge with B. elliptica on lily leaves. Similar expression profiles of homologues of LfGRP1, LfMT1, and LfPsbR (named LsGRP1, LsMT1, and LsPsbR, respectively) were presented in Lilium oriental hybrid Star Gazer. In addition, application of the photosynthetic inhibitor, 3-(3,4-dichlorophenyl)-1,1-dimethylurea, on the leaves reduced disease severity and expressions of LsGRP1 and LsPsbR just as that in response to B. cereus C1L treatment.

Physiological response of Bacillus cereus C1L-induced systemic resistance in lily against Botrytis leaf blight

Bacillus cereus C1L has been demonstrated to induce systemic disease resistance against Botrytis elliptica in lily. The objective of this study was to investigate physiological responses of B. cereus C1L-triggered systemic resistance in lily cv. Star Gazer against B. elliptica. By histological and biochemical analyses, leaves inoculated with B. elliptica displayed cell death, H2O2 accumulation and lignin deposition. As plants were elicited with B. cereus C1L, cell death, H2O2 accumulation and lignin deposition in leaves caused by B. elliptica infection were suppressed, revealing that suppression of oxidative burst might be associated with B. cereus C1L-induced systemic resistance. In reactive oxygen species inhibitors assays, B. elliptica-caused lesion numbers and H2O2 accumulation in lily leaves were significantly reduced as leaves were pretreated with catalase or diphenylene iodonium. Furthermore, the expression of LsGRP1 and LsPsbR in leaves elicited with B. cereus C1L and inoculated with B. elliptica was decreased. The same expression pattern was also observed in leaves pretreated with catalase or diphenylene iodonium and inoculated with B. elliptica. These results suggest that B. cereus C1L-induced systemic resistance may be related to suppression or alleviation of oxidative stress and cell death of lily caused by B. elliptica.

Characterization of the Dual Subcellular Localization of Lilium LsGRP1, a Plant Class II Glycine-Rich Protein

The defense-related gene LsGRP1 exhibits an increased level of expression in Lilium spp. after being infected by Botrytis elliptica, the fungal pathogen of lily leaf blight. In this study, the expression profile of the LsGRP1 protein (a plant class II glycine-rich protein) was characterized biochemically and its subcellular localization in lily leaves was evaluated using immunohistochemistry, enhanced green fluorescent protein (EGFP) imaging, and protein extraction analysis. Using an LsGRP1-specific antibody, LsGRP1 was found to be most abundant in epidermal cells and phloem tissues. Leaves from lily plants at different growth stages demonstrated similar levels of 14- and 16-kDa LsGRP1 and a decreased amount of 23-kDa LsGRP1 at the senescence stage. LsGRP1-EGFP imaging and protein extraction assays revealed that 14-kDa LsGRP1 was located in the plasma membrane whereas 16- and 23-kDa LsGRP1 was weakly bound to the cell wall. The time course analyses of LsGRP1 expression in response to salicylic acid treatment or B. elliptica infection showed an increased accumulation of 14- and 23-kDa LsGRP1 over time. Because 23-kDa LsGRP1 could be detected by an ubiquitin antibody, conversion of 14-kDa to 23-kDa LsGRP1 via mono-ubiquitination was presumed, which is a phenomenon that has not been reported for a plant class II glycine-rich protein.

Expression and functional characterization of the Agrobacterium VirB2 amino acid substitution variants in T-pilus biogenesis, virulence, and transient transformation efficiency.

Agrobacterium tumefaciens is a phytopathogenic bacterium that causes crown gall disease by transferring transferred DNA (T-DNA) into the plant genome. The translocation process is mediated by the type IV secretion system (T4SS) consisting of the VirD4 coupling protein and 11 VirB proteins (VirB1 to VirB11). All VirB proteins are required for the production of T-pilus, which consists of processed VirB2 (T-pilin) and VirB5 as major and minor subunits, respectively. VirB2 is an essential component of T4SS, but the roles of VirB2 and the assembled T-pilus in Agrobacterium virulence and the T-DNA transfer process remain unknown. Here, we generated 34 VirB2 amino acid substitution variants to study the functions of VirB2 involved in VirB2 stability, extracellular VirB2/T-pilus production and virulence of A. tumefaciens. From the capacity for extracellular VirB2 production (ExB2+ or ExB2−) and tumorigenesis on tomato stems (Vir+ or Vir−), the mutants could be classified into three groups: ExB2−/Vir−, ExB2−/Vir+, and ExB2+/Vir+. We also confirmed by electron microscopy that five ExB2−/Vir+ mutants exhibited a wild-type level of virulence with their deficiency in T-pilus formation. Interestingly, although the five T-pilus−/Vir+ uncoupling mutants retained a wild-type level of tumorigenesis efficiency on tomato stems and/or potato tuber discs, their transient transformation efficiency in Arabidopsis seedlings was highly attenuated. In conclusion, we have provided evidence for a role of T-pilus in Agrobacterium transformation process and have identified the domains and amino acid residues critical for VirB2 stability, T-pilus biogenesis, tumorigenesis, and transient transformation efficiency.