Pseudomonas Syringae Pv Phaseolicola Research Articles

English

Fungal endophytes are regarded as inexhaustible sources of pharmaceuticals and agrochemicals with profound antibacterial, anticancer or antifungal activities. Diaporthe sp., an endophytic fungus residing in medicinal plant S. cordatum, showed a good antagonism against bacterial pathogens of beans; Pseudomonas syringae pv phaseolicola (Psp) and Xanthomonas axonopodis pv phaseoli (Xap), with zones of inhibitions of 14.00 ± 1.15 and 17.00 ± 0.58 mm against the test organisms respectively. Large scale fermentation of Diaporthe sp. was performed on rice media after which ultrasonic extraction on methanol was done to yield methanol crude extract. Methanol crude extract was then partitioned between hexane and ethyl acetate to yield their respective crude extracts. Ethyl acetate fraction of Diaporthe sp. yielded one new naphthalene derivative compound which was accorded IUPAC name as 3-methoxy-5-methylnaphthalene-1, 7-diol after series of purifications on column chromatography as well as preparative high-performance liquid chromatography (pHPLC). Structure determination of isolated compounds was performed on 1D and 2D Nuclear Magnetic Resonance (NMR) spectroscopy experiments as well as a mass spectrometer to affirm its molecular mass. The F3 that yielded compound 1 had palpable antibacterial activities against Psp and Xap, with corresponding Minimum Inhibitory Concentration (MIC) values of 2.50 mg/ml (7.00 ± 0.00 mm) and 1.25 mg/ml (7.67 ± 0.33 mm) against the tests organisms respectively. These slight MIC values are chiefly attributed to the presence of active secondary metabolites in the fungal extracts that act against the test pathogens. This has therefore confirmed that fungal endophytes and their extractives have desirable antibacterial activities hence can be used in the formulation of agrochemicals or used as bio-control agents in crop protection especially in common beans (Phaseolus vulgaris L). Key words: Endophytes, Diaporthe sp, bean bacterial pathogens.

STRUVITE PRODUCTION BY PSEUDOMONAS SYRINGAE PV PHASEOLICOLA

Struvite is a biogenic mineral of low solubility. For many years, it has been considered as a fertilizer, but due to the additional cost of manufacture, its use has been limited to only high-value crops. Struvite production by some bacterial strains have been previously reported. However, this is the first study that reports struvite production in Pseudomonas syringae pv phaseolicola strain. Crystal formation was observed within four days of incubation on solid media. Microscopy, X-ray diffraction, Scanning electron microscope and Energy dispersive x-ray spectroscopy analysis confirmed the crystal structure as Struvite. Moreover, this study suggests a possible biotechnological use of P. syringae pv phaseolicola for struvite production for agricultural applications.

The Pseudomonas syringae pv. tomato Type III Effector HopM1 Suppresses Arabidopsis Defenses Independent of Suppressing Salicylic Acid Signaling and of Targeting AtMIN7

Pseudomonas syringae pv tomato strain DC3000 (Pto) delivers several effector proteins promoting virulence, including HopM1, into plant cells via type III secretion. HopM1 contributes to full virulence of Pto by inducing degradation of Arabidopsis proteins, including AtMIN7, an ADP ribosylation factor-guanine nucleotide exchange factor. Pseudomonas syringae pv phaseolicola strain NPS3121 (Pph) lacks a functional HopM1 and elicits robust defenses in Arabidopsis thaliana, including accumulation of pathogenesis related 1 (PR-1) protein and deposition of callose-containing cell wall fortifications. We have examined the effects of heterologously expressed HopM1Pto on Pph-induced defenses. HopM1 suppresses Pph-induced PR-1 expression, a widely used marker for salicylic acid (SA) signaling and systemic acquired resistance. Surprisingly, HopM1 reduces PR-1 expression without affecting SA accumulation and also suppresses the low levels of PR-1 expression apparent in SA-signaling deficient plants. Further, HopM1 enhances the growth of Pto in SA-signaling deficient plants. AtMIN7 contributes to Pph-induced PR-1 expression. However, HopM1 fails to degrade AtMIN7 during Pph infection and suppresses Pph-induced PR-1 expression and callose deposition in wild-type and atmin7 plants. We also show that the HopM1-mediated suppression of PR-1 expression is not observed in plants lacking the TGA transcription factor, TGA3. Our data indicate that HopM1 promotes bacterial virulence independent of suppressing SA-signaling and links TGA3, AtMIN7, and other HopM1 targets to pathways distinct from the canonical SA-signaling pathway contributing to PR-1 expression and callose deposition. Thus, efforts to understand this key effector must consider multiple targets and unexpected outputs of its action.

Phosphorylation of HopQ1, a Type III Effector from Pseudomonas syringae, Creates a Binding Site for Host 14-3-3 Proteins

HopQ1 (for Hrp outer protein Q), a type III effector secreted by Pseudomonas syringae pv phaseolicola, is widely conserved among diverse genera of plant bacteria. It promotes the development of halo blight in common bean (Phaseolus vulgaris). However, when this same effector is injected into Nicotiana benthamiana cells, it is recognized by the immune system and prevents infection. Although the ability to synthesize HopQ1 determines host specificity, the role it plays inside plant cells remains unexplored. Following transient expression in planta, HopQ1 was shown to copurify with host 14-3-3 proteins. The physical interaction between HopQ1 and 14-3-3a was confirmed in planta using the fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy technique. Moreover, mass spectrometric analyses detected specific phosphorylation of the canonical 14-3-3 binding site (RSXpSXP, where pS denotes phosphoserine) located in the amino-terminal region of HopQ1. Amino acid substitution within this motif abrogated the association and led to altered subcellular localization of HopQ1. In addition, the mutated HopQ1 protein showed reduced stability in planta. These data suggest that the association between host 14-3-3 proteins and HopQ1 is important for modulating the properties of this bacterial effector.

Determination of responses of different bean cultivars against races of Pseudomonas syringae pv phaseolicola, causal agent of halo blight of bean

Use of resistant plant varieties combined with other disease management practices is regarded as the most practical approach to control of seed-borne bacterial disease agents. In this study, responses of different bean cultivars to nine different races of Pseudomonas syringae pv phaseolicola, the causal agent of bacterial halo blight of common bean (Phaseolus vulgaris L.), were determined. During compatible interaction in susceptible cultivars, virulent bacterial races caused water soaked lesion at sites of inoculation. Similar lesions developed in moderately resistant cultivars but symptoms were later associated with more tissue browning around the sites of inoculation. In contrast, the resistant response, produced the characteristic hypersensitive reaction (HR), was characterized as a small discrete browning and tissue collapse at site of inoculation. No local cultivars showed complete resistance to all races tested. Bean cultivars Sehirali-90 and Goynuk-98 were found to be resistant or moderately resistant to five different bacterial races. Bean cultivar, Karacasehir-90, on the other hand, was found to be resistant or moderately resistant to six different bacterial races. Analysis of bacterial growth and the accumulation of isoflavonoid bean phytoalexin, phaseollin in planta were carried out for tissues expressing compatible and incompatible interactions to enable a link to be made between reaction phenotypes and restriction of bacterial growth and phytoalexin accumulations. Development of the HR was clearly associated with the restricted multiplication of bacteria during incompatible interactions. A time-course accumulation analysis on pods treated with different races of bacterial agent showed that a strong correlation was observed between the timing and extent of cell death and accumulation of phaseollin, being rapid and extensive in incompatible interactions compared to compatible interaction.

Evidence for a Coiled-coil Interaction Mode of Disordered Proteins from Bacterial Type III Secretion Systems

Gene clusters encoding various type III secretion system (T3SS) injectisomes, frequently code downstream of the conserved atpase gene for small hydrophilic proteins whose amino acid sequences display a propensity for intrinsic disorder and coiled-coil formation. These properties were confirmed experimentally for a member of this class, the HrpO protein from the T3SS of Pseudomonas syringae pv phaseolicola: HrpO exhibits high alpha-helical content with coiled-coil characteristics, strikingly low melting temperature, structural properties that are typical for disordered proteins, and a pronounced self-association propensity, most likely via coiled-coil interactions, resulting in heterogeneous populations of quaternary complexes. HrpO interacts in vivo with HrpE, a T3SS protein for which coiled-coil formation is also strongly predicted. Evidence from HrpO analogues from all T3SS families and the flagellum suggests that the extreme flexibility and propensity for coiled-coil interactions of this diverse class of small, intrinsically disordered proteins, whose structures may alter as they bind to their cognate folded protein targets, might be important elements in the establishment of protein-protein interaction networks required for T3SS function.

A Genome-Wide Functional Investigation into the Roles of Receptor-Like Proteins in Arabidopsis

Receptor-like proteins (RLPs) are cell surface receptors that typically consist of an extracellular leucine-rich repeat domain, a transmembrane domain, and a short cytoplasmatic tail. In several plant species, RLPs have been found to play a role in disease resistance, such as the tomato (Solanum lycopersicum) Cf and Ve proteins and the apple (Malus domestica) HcrVf2 protein that mediate resistance against the fungal pathogens Cladosporium fulvum, Verticillium spp., and Venturia inaequalis, respectively. In addition, RLPs play a role in plant development; Arabidopsis (Arabidopsis thaliana) TOO MANY MOUTHS (TMM) regulates stomatal distribution, while Arabidopsis CLAVATA2 (CLV2) and its functional maize (Zea mays) ortholog FASCINATED EAR2 regulate meristem maintenance. In total, 57 RLP genes have been identified in the Arabidopsis genome and a genome-wide collection of T-DNA insertion lines was assembled. This collection was functionally analyzed with respect to plant growth and development and sensitivity to various stress responses, including susceptibility toward pathogens. A number of novel developmental phenotypes were revealed for our CLV2 and TMM insertion mutants. In addition, one AtRLP gene was found to mediate abscisic acid sensitivity and another AtRLP gene was found to influence nonhost resistance toward Pseudomonas syringae pv phaseolicola. This genome-wide collection of Arabidopsis RLP gene T-DNA insertion mutants provides a tool for future investigations into the biological roles of RLPs.

The outcomes of concentration-specific interactions between salicylate and jasmonate signaling include synergy, antagonism, and oxidative stress leading to cell death.

Salicylic acid (SA) has been proposed to antagonize jasmonic acid (JA) biosynthesis and signaling. We report, however, that in salicylate hydroxylase-expressing tobacco (Nicotiana tabacum) plants, where SA levels were reduced, JA levels were not elevated during a hypersensitive response elicited by Pseudomonas syringae pv phaseolicola. The effects of cotreatment with various concentrations of SA and JA were assessed in tobacco and Arabidopsis (Arabidopsis thaliana). These suggested that there was a transient synergistic enhancement in the expression of genes associated with either JA (PDF1.2 [defensin] and Thi1.2 [thionin]) or SA (PR1 [PR1a-beta-glucuronidase in tobacco]) signaling when both signals were applied at low (typically 10-100 microm) concentrations. Antagonism was observed at more prolonged treatment times or at higher concentrations. Similar results were also observed when adding the JA precursor, alpha-linolenic acid with SA. Synergic effects on gene expression and plant stress were NPR1- and COI1-dependent, SA- and JA-signaling components, respectively. Electrolyte leakage and Evans blue staining indicated that application of higher concentrations of SA + JA induced plant stress or death and elicited the generation of apoplastic reactive oxygen species. This was indicated by enhancement of hydrogen peroxide-responsive AoPR10-beta-glucuronidase expression, suppression of plant stress/death using catalase, and direct hydrogen peroxide measurements. Our data suggests that the outcomes of JA-SA interactions could be tailored to pathogen/pest attack by the relative concentration of each hormone.

Localization of hydrogen peroxide accumulation during the hypersensitive reaction of lettuce cells to Pseudomonas syringae pv phaseolicola.

The active oxygen species hydrogen peroxide (H2O2) was detected cytochemically by its reaction with cerium chloride to produce electron-dense deposits of cerium perhydroxides. In uninoculated lettuce leaves, H2O2 was typically present within the secondary thickened walls of xylem vessels. Inoculation with wild-type cells of Pseudomonas syringae pv phaseolicola caused a rapid hypersensitive reaction (HR) during which highly localized accumulation of H2O2 was found in plant cell walls adjacent to attached bacteria. Quantitative analysis indicated a prolonged burst of H2O2 occurring between 5 to 8 hr after inoculation in cells undergoing the HR during this example of non-host resistance. Cell wall alterations and papilla deposition, which occurred in response to both the wild-type strain and a nonpathogenic hrpD mutant, were not associated with intense staining for H2O2, unless the responding cell was undergoing the HR. Catalase treatment to decompose H2O2 almost entirely eliminated staining, but 3-amino-1,2,4-triazole (catalase inhibitor) did not affect the pattern of distribution of H2O2 detected. H2O2 production was reduced more by the inhibition of plant peroxidases (with potassium cyanide and sodium azide) than by inhibition of neutrophil-like NADPH oxidase (with diphenylene iodonium chloride). Results suggest that CeCl3 reacts with excess H2O2 that is not rapidly metabolized during cross-linking reactions occurring in cell walls; such an excess of H2O2 in the early stages of the plant-bacterium interaction was only produced during the HR. The highly localized accumulation of H2O2 is consistent with its direct role as an antimicrobial agent and as the cause of localized membrane damage at sites of bacterial attachment.

Localization of Hydrogen Peroxide Accumulation during the Hypersensitive Reaction of Lettuce Cells to Pseudomonas syringae pv phaseolicola

Localization of Hydrogen Peroxide Accumulation during the Hypersensitive Reaction of Lettuce Cells to Pseudomonas syringae pv phaseolicola

A simple and efficient PCR method for the specific detection of Pseudomonas syringae pv phaseolicola in bean seeds

Using Southern blot hybridizations, it was found that the gene encoding the phaseolotoxin-insensitive ornithyl carbamoyl transferase (argK) was specific for Pseudomonas syringae pv. phaseolicola, the causal agent of the halo-blight disease. Based on these findings, a PCR protocol was developed for the specific detection of P.syringae. pv. phaseolicola in water-extracts of soaked bean seed. For this PCR protocol, two oligonucleotide primers were designed, based on the sequence of argK, which allowed the detection of a specific 1kb fragment. The protocol is simple since PCR was directly applied to bacterial suspensions, thus avoiding DNA extraction. The sensitivity of detection was increased by allowing the bacteria present in seed extracts to multiply in semi-selective media for 18h prior to PCR amplification. The detection threshold by visual detection using ethidium bromide staining was one naturally infected seed in lots of 400 to 600 seeds.

Hrp Mutant of Pseudomonas syringae pv phaseolicola Induces Cell Wall Alterations but Not Membrane Damage Leading to the Hypersensitive Reaction in Lettuce.

Both wild-type (S21-WT) and hrpD- (S21-533) strains of Pseudomonas syringae pv phaseolicola induced the formation of large paramural papillae in lettuce (Lactuca sativa) mesophyll cells adjacent to bacterial colonies. Localized alterations to the plant cell wall included deposition of hydroxyproline-rich glycoproteins, phe-nolics, and callose, and were associated with proliferation of the endoplasmic reticulum and multivesicular bodies. Tissue collapse during the hypersensitive reaction caused by S21-WT was associated with electrolyte leakage and rapid accumulation of the phy-toalexin lettucenin A, both of which followed membrane damage indicated by the failure of mesophyll cells to plasmolyze. A few cells lost the ability to plasmolyze after inoculation with S21-533, and low levels of lettucenin A were recorded, but neither leakage of electrolytes nor tissue collapse were detected. Dysfunction of the plasma membrane in cells adjacent to colonies of S21-WT led to extensive vacuolation of the cytoplasm, organelle disruption, and cytoplasmic collapse[mdash]changes unlike those occurring in cells undergoing apoptosis. Strain S21-533 remained viable within symptomless tissue, whereas cells of S21-WT were killed as a consequence of the hypersensitive reaction. Our observations emphasize the subtle coordination of the plant's response occurring at the subcellular level.

Volatile Products of the Lipoxygenase Pathway Evolved from Phaseolus vulgaris (L.) Leaves Inoculated with Pseudomonas syringae pv phaseolicola.

Activation of the "lipoxygenase pathway" in plants gives rise to a series of products derived from fatty acids. Analysis by gas chromatography-mass spectroscopy of volatile products produced by Phaseolus vulgaris (L.) cv Red Mexican leaves during a hypersensitive resistance response (HR) to the plant pathogenic bacterium Pseudomonas syringae pv phaseolicola showed evolution of several lipid-derived volatiles, including cis-3-hexenol and trans-2-hexenal, which arise from the 13-hydroperoxide of linolenic acid. These compounds were not produced in detectable amounts by buffer-inoculated leaves, nor did they evolve to such a high degree during comparable stages of the susceptible response. The absence of trans-2,cis-6-nonadienal, a product expected from 9-hydroperoxide of linolenic acid, suggests that lipid peroxidation during the HR proceeded primarily enzymically via bean lipoxygenase, which produces the 13-hydroperoxide, and not via autoxidative processes. The effects of trans-2-hexenal, cis-3-hexenol, and traumatic acid on P.s pv phaseolicola were investigaed. trans-2-Hexenal appeared to be highly bactericidal at low concentrations, whereas cis-3-hexenol was bactericidal only at much higher concentrations. Traumatic acid appeared to have no effect on P.s. pv. phaseolicola at the concentrations tested. These results demonstrate that during plant defense responses against microbial attack, several lipid-derived compounds are produced by the plant, some of which possess antimicrobial activity and conceivably are involved in plant disease resistance. The time of production of these substances, in amounts that would be expected to be antibacterial in vitro, correlated with a slowing down of the growth rate of bacteria in the leaves and was seen at a time before the accumulation of isoflavonoid phytoalexins in the host.

Suppression of Bean Defense Responses by Pseudomonas syringae.

We have developed a model system to examine suppression of defense responses in bean by the compatible bacterium Pseudomonas syringae pv phaseolicola. Previously, we have shown that there is a general mechanism for the induction of the bean defense genes phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), and chitinase (CHT) by incompatible, compatible, and nonpathogenic bacteria. Here, we show that bean plants infiltrated with isolates of P. s. phaseolicola failed to produce transcripts for PAL, CHS, or CHI up to 120 hr after infiltration and CHT transcript accumulation was significantly delayed when compared to the incompatible P. syringae strains. Infiltration of bean plants with 108 cells per mL of P. s. phaseolicola NPS3121 8 hr prior to infiltration with an equal concentration of incompatible P. s. pv tabaci Pt11528 significantly reduced the typical profile of defense transcript accumulation when compared to plants infiltrated with Pt11528 alone. A corresponding suppression of phytoalexin accumulation was also observed. NPS3121 also suppressed PAL, CHS, CHI, and CHT transcript accumulation and phytoalexin production induced by Escherichia coli DH5[alpha] or the elicitor glutathione. Heat-killed NPS3121 cells or cells treated with protein synthesis inhibitors lost the suppressor activity. Taken together, these experiments suggest that NPS3121 has an active mechanism to suppress the accumulation of defense transcripts and phytoalexin biosynthesis in bean.

‘Montecarlo’ and ‘Monterosa’, ‘Borlotto’-type Dry Beans

Abstract ‘Montecarlo’ (P288) and ‘Monterosa’ (P358) are ‘Borlotto’-type dry beans with determinate type I plant habit (Singh, 1982) and resistance to bean common mosaic virus (BCMV) and halo blight [Pseudomonas syringae pv phaseolicola (Burk.) Dows.] (Hill et al., 1972; Drifjhout et al., 1978). Both were developed at the Experimental Institute for Vegetable Crops, Montanaso Lombardo, Italy. ‘Borlotto’ is a shell bean traditionally grown in Italy for direct marketing of ripe pods and dry seeds or for processing. ‘Borlotto’ has seeds with red stripes on a beige background at maturity and is similar to ‘Taylor's Horticultural’ (Asgrow Seed).

Association between Symptom Development and Inhibition of Ornithine Carbamoyltransferase in Bean Leaves Treated with Phaseolotoxin

The chlorosis symptom that characterizes the halo blight disease of Phaseolus vulgaris L. is caused by phaseolotoxin produced by the plant pathogenic bacterium Pseudomonas syringae pv phaseolicola. Phaseolotoxin is hydrolyzed by plant peptidases to N(delta)(N'-sulpho-diaminophosphinyl) -l-ornithine which also causes chlorosis and is reported to be an irreversible inhibitor of ornithine carbamoyltransferase (OCTase). We have examined the hypothesis that inhibition of OCTase is the primary action of phaseolotoxin that leads to chlorosis.Chlorotic spots appeared on the primary leaves of P. vulgaris seedlings during the 2 days following leaf prick application of a minimum of 30 picomole phaseolotoxin. OCTase in extracts of the lesions was reduced to 20%, or less, of the activity in controls. Four hours after the application of phaseolotoxin the concentration of free ornithine increased more than 2-fold. Other amino acids, especially glutamine and asparagine-but not arginine-increased later. Chlorophyll remained at a constant level in the phaseolotoxin-treated tissue and the appearance of chlorosis was due to the increase in chlorophyll in the surrounding unaffected tissue.Clear halo symptoms developed only on primary leaves of the youngest seedlings (treated 6-7 days after germination). Lesions did not develop on primary leaves of seedlings more than 14 days old, in which the chlorophyll concentration had reached a maximum. OCTase also was inhibited in the symptomless tissue from older leaves treated with phaseolotoxin, but there was no accumulation of amino acids, including ornithine. A single appliction of 200 nanomoles arginine resulted in the complete regreening of the chlorosis caused by phaseolotoxin. Soluble protein was lower in the chlorotic tissue than in the controls, but increased to greater than the control value following the appliction of arginine. These results suggest that phaseolotoxin-induced chlorosis results from reduced chlorophyll synthesis that is associated with arginine-starvation in the tissue where OCTase is inhibited.