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Plant root-associated bacteria (rhizobacteria) elicit plant basal immunity referred to as induced systemic resistance (ISR) against multiple pathogens. Among multi-bacterial determinants involving such ISR, the induction of ISR and promotion of growth by bacterial volatile compounds was previously reported. To exploit global de novo expression of plant proteins by bacterial volatiles, proteomic analysis was performed after exposure of Arabidopsis plants to the rhizobacterium Bacillus subtilis GB03. Ethylene biosynthesis enzymes were significantly up-regulated. Analysis by quantitative reverse transcriptase polymerase chain reaction confirmed that ethylene biosynthesis-related genes SAM-2, ACS4, ACS12, and ACO2 as well as ethylene response genes, ERF1, GST2, and CHIB were up-regulated by the exposure to bacterial volatiles. More interestingly, the emission of bacterial volatiles significantly up-regulated both key defense mechanisms mediated by jasmonic acid and salicylic acid signaling pathways. In addition, high accumulation of antioxidant proteins also provided evidence of decreased sensitivity to reactive oxygen species during the elicitation of ISR by bacterial volatiles. The present results suggest that the proteomic analysis of plant defense responses in bacterial volatile-mediated ISR can reveal the mechanisms of plant basal defenses orchestrated by endogenous ethylene production pathways and the generation of reactive oxygen species.

There is considerable interest in redox regulation and new targets for thioredoxin and glutaredoxin are now being identified. It would be of great benefit to the field to have a list of all possible candidates for redox regulation--that is all disulfide proteins in plant. We developed a simple and very powerful method for identifying proteins with disulfide bonds in vivo. In this method, free thiols in proteins are fully blocked by alkylation, following which disulfide cysteines are converted to sulfhydryl groups by reduction. Finally, proteins with sulfhydryls are isolated by thiol affinity chromatography. Our method is unique in that membrane proteins as well as water-soluble proteins are examined for their disulfide nature. By applying this method to Arabidopsis thaliana we identified 65 putative disulfide proteins, including 20 that had not previously been demonstrated to be regulated by redox state. The newly identified, possibly redox-regulated proteins include: violaxanthin de-epoxidase, two oxygen-evolving enhancer proteins, carbonic anhydrase, photosystem I reaction center subunit N, photosystem I subunit III, S-adenosyl-L-methionine carboxyl methyltransferase, guanylate kinase, and bacterial mutT homolog. Possible functions of disulfide bonding in these proteins are discussed.

SummaryAgrobacterium tumefaciens causes crown gall disease. Although Agrobacterium can be popularly used for genetic engineering, the influence of aboveground insect infestation on Agrobacterium induced gall formation has not been investigated.Nicotiana benthamiana leaves were exposed to a sucking insect (whitefly) infestation and benzothiadiazole (BTH) for 7 d, and these exposed plants were inoculated with a tumorigenic Agrobacterium strain. We evaluated, both in planta and in vitro, how whitefly infestation affects crown gall disease.Whitefly-infested plants exhibited at least a two-fold reduction in gall formation on both stem and crown root. Silencing of isochorismate synthase 1 (ICS1), required for salicylic acid (SA) synthesis, compromised gall formation indicating an involvement of SA in whiteflyderived plant defence against Agrobacterium. Endogenous SA content was augmented in whitefly-infested plants upon Agrobacterium inoculation. In addition, SA concentration was three times higher in root exudates from whitefly-infested plants. As a consequence, Agrobacterium-mediated transformation of roots of whitefly-infested plants was clearly inhibited when compared to control plants. These results suggest that aboveground whitefly infestation elicits systemic defence responses throughout the plant.Our findings provide new insights into insect-mediated leaf-root intra-communication and a framework to understand interactions between three organisms: whitefly, N. benthamiana and Agrobacterium.

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