High throughput virus-induced gene silencing implicates heat shock protein 90 in plant disease resistance.
(9/851)
Virus-induced gene silencing was used to assess the function of random Nicotiana benthamiana cDNAs in disease resistance. Out of 4992 cDNAs tested from a normalized library, there were 79 that suppressed a hypersensitive response (HR) associated with Pto-mediated resistance against Pseudomonas syringae. However, only six of these clones blocked the Pto-mediated suppression of P.syringae growth. The three clones giving the strongest loss of Pto resistance had inserts corresponding to HSP90 and also caused loss of Rx-mediated resistance against potato virus X and N-mediated tobacco mosaic virus resistance. The role of HSP90 as a cofactor of disease resistance is associated with stabilization of Rx protein levels and could be accounted for in part by SGT1 and other cofactors of disease resistance acting as co-chaperones. This approach illustrates the potential benefits and limitations of RNA silencing in forward screens of gene function in plants. (+info)
Flagellin glycosylation island in Pseudomonas syringae pv. glycinea and its role in host specificity.
(10/851)
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. (+info)
Nitric oxide does not trigger early programmed cell death events but may contribute to cell-to-cell signaling governing progression of the Arabidopsis hypersensitive response.
(11/851)
Nitric oxide (NO) has been suggested to play a role in the hypersensitive response (HR). Single- and double-label fluorescence microscopy experiments were conducted using Arabidopsis leaves infected with Pseudomonas syringae pv. tomato DC3000 carrying either avrB or avrRpt2. Kinetics of NO production were followed by measurement of green 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM) triazole fluorescence in leaves coinfiltrated with DAF-FM diacetate. Kinetics of hypersensitive cell death were followed by measurement of cytoplasmic red fluorescence following internalization of coinfiltrated propidium iodide through compromised plasma membranes. Neither NO accumulation nor cell death was seen until approximately 3 h postinoculation of Columbia leaves with DC3000.avrB or approximately 5.5 h post-inoculation with DC3000.avrRpt2. Subsequent NO accumulation kinetics closely paralleled HR progression in both Columbia and ndr1-1 mutant plants. These data established that NO accumulation does not happen sufficiently early for NO to be a signaling component controlling HR triggering. NO accumulation did contribute to the HR, as proven by an approximately 1-h delay in cell death kinetics caused by an NO scavenger or an NO synthase inhibitor. NO was first seen as punctate foci at the cell surface. Subsequent NO accumulation patterns were consistent with NO being an intercellular signal that functions in cell-to-cell spread of the HR. (+info)
GacA, the response regulator of a two-component system, acts as a master regulator in Pseudomonas syringae pv. tomato DC3000 by controlling regulatory RNA, transcriptional activators, and alternate sigma factors.
(12/851)
Concerted investigations of factors affecting host-pathogen interactions are now possible with the model plant Arabidopsis thaliana and its model pathogen Pseudomonas syringae pv. tomato DC3000, as their whole genome sequences have become available. As a prelude to analysis of the regulatory genes and their targets, we have focused on GacA, the response regulator of a two-component system. The DC3000 gene was cloned by testing for the reversal of phenotypes of an Erwinia GacA- mutant. A GacA- mutant of DC3000 constructed by marker exchange produces much-reduced levels of transcripts of three alternate sigma factors: HrpL, required for the production of effector proteins and their translocation via the type III secretion system; RpoS, required for stress responses and secondary metabolite production; and RpoN, required for an assortment of metabolic processes and expression of hrpL. GacA deficiency also reduces the expression of hrpR and hrpS, which specify enhancer-binding proteins of the NtrC family required for hrpL transcription; ahlI and ahlR, the genes for quorum sensing signal; salA, a regulatory gene known to control virulence; CorS, a sensor kinase; CorR, the cognate response regulator that controls coronatine biosynthetic genes; and rsmB and rsmZ, which specify untranslatable regulatory RNA species. gacA expression itself is regulated by environmental conditions in DC3000, since transcript levels are affected by growth phase and media composition. The observations that high levels of gacA RNA occur in the hrp-inducing medium and GacA deficiency reduces the levels of rpoS expression implicate an important role of GacA in stress responses of DC3000. Consistent with the effects on hrpL expression, the GacA- mutant produces lower levels of transcripts of avr, hrp, and hop genes controlled by HrpL. In addition, GacA deficiency results in reduced levels of transcripts of several HrpL-independent genes. As would be expected, these effects on gene expression cause drastic changes in bacterial behavior: virulence towards A. thaliana and tomato; multiplication in planta; efficiency of the induction of the hypersensitive reaction (HR); production of pigment and N-acyl-homoserine lactone (AHL), the presumed quorum-sensing signal; and swarming motility. Our findings establish that GacA, located at the top in a regulatory cascade in DC3000, functions as a central regulator by controlling an assortment of transcriptional and posttranscriptional factors. (+info)
Characterization of the argA gene required for arginine biosynthesis and syringomycin production by Pseudomonas syringae pv. syringae.
(13/851)
Two types of necrosis-inducing lipodepsipeptide toxins, called syringomycin and syringopeptin, are major virulence factors of Pseudomonas syringae pv. syringae strain B301D. A previous study showed that a locus, called syrA, was required for both syringomycin production and plant pathogenicity, and the syrA locus was speculated to encode a regulator of toxin production. In this study, sequence analysis of the 8-kb genomic DNA fragment that complements the syrA phenotype revealed high conservation among a broad spectrum of fluorescent pseudomonads. The putative protein encoded by open reading frame 4 (ORF4) (1,299 bp) in the syrA locus region exhibited 85% identity to ArgA, which is involved in arginine biosynthesis in Pseudomonas aeruginosa. Growth of strain W4S2545, the syrA mutant, required supplementation of N minimal medium with arginine. Similarly, syringomycin production of syrA mutant W4S2545 was restored by the addition of arginine to culture media. Furthermore, the insertion of Tn5 in the genome of the syrA mutant W4S2545 was localized between nucleotides 146 and 147 in ORF4, and syringomycin production was complemented in trans with the wild-type DNA fragment containing intact ORF4. These results demonstrate that the syrA locus is the argA gene of P. syringae pv. syringae and that argA is directly involved in arginine biosynthesis and therefore indirectly affects syringomycin production because of arginine deficiency. (+info)
Concomitant induction of systemic resistance to Pseudomonas syringae pv. lachrymans in cucumber by Trichoderma asperellum (T-203) and accumulation of phytoalexins.
(14/851)
Most studies on the reduction of disease incidence in soil treated with Trichoderma asperellum have focused on microbial interactions rather than on plant responses. This study presents conclusive evidence for the induction of a systemic response against angular leaf spot of cucumber (Pseudomonas syringae pv. lachrymans) following application of T. asperellum to the root system. To ascertain that T. asperellum was the only microorganism present in the root milieu, plants were grown in an aseptic hydroponic growth system. Disease symptoms were reduced by as much as 80%, corresponding to a reduction of 2 orders of magnitude in bacterial cell densities in leaves of plants pretreated with T. asperellum. As revealed by electron microscopy, bacterial cell proliferation in these plants was halted. The protection afforded by the biocontrol agent was associated with the accumulation of mRNA of two defense genes: the phenylpropanoid pathway gene encoding phenylalanine ammonia lyase (PAL) and the lipoxygenase pathway gene encoding hydroxyperoxide lyase (HPL). This was further supported by the accumulation of secondary metabolites of a phenolic nature that showed an increase of up to sixfold in inhibition capacity of bacterial growth in vitro. The bulk of the antimicrobial activity was found in the acid-hydrolyzed extract containing the phenolics in their aglycone form. High-performance liquid chromatography analysis of phenolic compounds showed a marked change in their profile in the challenged, preelicited plants relative to that in challenged controls. The results suggest that similar to beneficial rhizobacteria, T. asperellum may activate separate metabolic pathways in cucumber that are involved in plant signaling and biosynthesis, eventually leading to the systemic accumulation of phytoalexins. (+info)
Differential survival of solitary and aggregated bacterial cells promotes aggregate formation on leaf surfaces.
(15/851)
The survival of individual Pseudomonas syringae cells was determined on bean leaf surfaces maintained under humid conditions or periodically exposed to desiccation stress. Cells of P. syringae strain B728a harboring a GFP marker gene were visualized by epifluorescence microscopy, either directly in situ or after recovery from leaves, and dead cells were identified as those that were stained with propidium iodide in such populations. Under moist, conducive conditions on plants, the proportion of total live cells was always high, irrespective of their aggregated state. In contrast, the proportion of the total cells that remained alive on leaves that were periodically exposed to desiccation stress decreased through time and was only approximately 15% after 5 days. However, the fraction of cells in large aggregates that were alive on such plants in both condition was much higher than more solitary cells. Immediately after inoculation, cells were randomly distributed over the leaf surface and no aggregates were observed. However, a very aggregated pattern of colonization was apparent within 7 days, and >90% of the living cells were located in aggregates of 100 cells or more. Our results strongly suggest that, although conducive conditions favor aggregate formation, such cells are much more capable of tolerating environmental stresses, and the preferential survival of cells in aggregates promotes a highly clustered spatial distribution of bacteria on leaf surfaces. (+info)
Characterization of CmaA, an adenylation-thiolation didomain enzyme involved in the biosynthesis of coronatine.
(16/851)
Several pathovars of Pseudomonas syringae produce the phytotoxin coronatine (COR), which contains an unusual amino acid, the 1-amino-2-ethylcyclopropane carboxylic acid called coronamic acid (CMA), which is covalently linked to a polyketide-derived carboxylic acid, coronafacic acid, by an amide bond. The region of the COR biosynthetic gene cluster proposed to be responsible for CMA biosynthesis was resequenced, and errors in previously deposited cmaA sequences were corrected. These efforts allowed overproduction of P. syringae pv. glycinea PG4180 CmaA in P. syringae pv. syringae FF5 as a FLAG-tagged protein and overproduction of P. syringae pv. tomato CmaA in Escherichia coli as a His-tagged protein; both proteins were in an enzymatically active form. Sequence analysis of CmaA indicated that there were two domains, an adenylation domain (A domain) and a thiolation domain (T domain). ATP-(32)PP(i) exchange assays showed that the A domain of CmaA catalyzes the conversion of branched-chain L-amino acids and ATP into the corresponding aminoacyl-AMP derivatives, with a kinetic preference for L-allo-isoleucine. Additional experiments demonstrated that the T domain of CmaA, which is posttranslationally modified with a 4'-phosphopantetheinyl group, reacts with the AMP derivative of L-allo-isoleucine to produce an aminoacyl thiolester intermediate. This covalent species was detected by incubating CmaA with ATP and L-[G-(3)H]allo-isoleucine, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. It is postulated that the L-allo-isoleucine covalently tethered to CmaA serves as the substrate for additional enzymes in the CMA biosynthetic pathway that catalyze cyclopropane ring formation, which is followed by thiolester hydrolysis, yielding free CMA. The availability of catalytically active CmaA should facilitate elucidation of the details of the subsequent steps in the formation of this novel cyclopropyl amino acid. (+info)