Protection of apple against fire blight induced by an hrpL mutant of Erwinia amylovora

A regulatory hrpL non-virulent mutant of Erwinia amylovora is effective in controlling fire blight disease when inoculated on apple seedlings simultaneously with the pathogenic parental strain. Mechanisms involved in this protective effect were investigated. The use of two marker genes, uidA and lacZ, expressed in the hrpL mutant and the pathogenic strain, respectively, allowed to localize simultaneously the two inoculated strains in plant tissue. An anti-β-glucuronidase antibody was also used to detect the hrpL mutant. Both techniques indicated that the two strains localized mainly in separate areas of the leaf tissue. In addition, leaves infiltrated with the hrpL mutant exhibited a significant increase in peroxidase activity in contrast to a hrp secretion mutant known to be less effective in the protection. It is suggested that protection obtained with the hrpL mutant relies on the physical separation between the mutant and the parental strain after co-inoculation and the rapid and sustained activation of plant defense mechanisms in reactive tissue, i.e. not invaded by the virulent strain.     

Erwinia amylovora modifies phenolic profiles of susceptible and resistant apple through its type III secretion system

Fire blight is a disease affecting Maloideae caused by the necrogenic bacterium Erwinia amylovora , which requires the type III protein secretion system (TTSS) for pathogenicity. Profiles of methanol-extractable leaf phenolics of two apple ( Malus  ×  domestica ) genotypes with contrasting susceptibility to this disease were analyzed by HPLC after infection. Some qualitative differences were recorded between the constitutive compositions of the two genotypes but in both of them dihydrochalcones accounted for more than 90% of total phenolics. Principal component analysis separated leaves inoculated with a virulent wild-type strain from those inoculated with a non-pathogenic TTSS-defective mutant or with water. The changes in levels of the various groups of phenolics in response to the virulent bacterium were similar between the two genotypes, with a significant decrease of dihydrochalcones and a significant increase of hydroxycinnamate derivatives. Differences between genotypes were, however, recorded in amplitude and kinetic of variation in these groups. Occurrence of oxidation and polymerization reactions is proposed, based on the browning process of infected tissues, but whether some by-products act in defense as toxic compounds remain to be tested. Among direct antibacterial constitutive compounds present in apple leaves, the dihydrochalcone phloretin only was found at levels close to lethal concentrations in both genotypes. However, E.   amylovora exhibited the ability to stabilize this compound at sublethal levels even in the resistant apple, rejecting the hypothesis of its involvement in the resistance of this genotype.     

HrpN of Erwinia amylovora functions in the translocation of DspA/E into plant cells

The type III secretion system (T3SS) is required by plant pathogenic bacteria for the translocation of certain bacterial proteins to the cytoplasm of plant cells or secretion of some proteins to the apoplast. The T3SS of Erwinia amylovora, which causes fire blight of pear, apple and other rosaceous plants, secretes DspA/E, which is an indispensable pathogenicity factor. Several other proteins, including HrpN, a critical virulence factor, are also secreted by the T3SS. Using a CyaA reporter system, we demonstrated that DspA/E is translocated into the cells of Nicotiana tabacum'Xanthi'. To determine if other T3-secreted proteins are needed for translocation of DspA/E, we examined its translocation in several mutants of E. amylovora strain Ea321. DspA/E was translocated by both hrpW and hrpK mutants, although with some delay, indicating that these two proteins are dispensable in the translocation of DspA/E. Remarkably, translocation of DspA/E was essentially abolished in both hrpN and hrpJ mutants; however, secretion of DspA/E into medium was not affected in any of the mentioned mutants. In contrast to the more virulent strain Ea273, secretion of HrpN was abolished in a hrpJ mutant of strain Ea321. In addition, HrpN was weakly translocated into plant cytoplasm. These results suggest that HrpN plays a significant role in the translocation of DspA/E, and HrpJ affects the translocation of DspA/E by affecting secretion or stability of HrpN. Taken together, these results explain the critical importance of HrpN and HrpJ to the development of fire blight.     

Pectobacterium carotovorum elicits plant cell death with DspE/F but the P. carotovorum DspE does not suppress callose or induce expression of plant genes early in plant-microbe interactions

The broad-host-range bacterial soft rot pathogen Pectobacterium carotovorum causes a DspE/F-dependent plant cell death on Nicotiana benthamiana within 24 h postinoculation (hpi) followed by leaf maceration within 48 hpi. P. carotovorum strains with mutations in type III secretion system (T3SS) regulatory and structural genes, including the dspE/F operon, did not cause hypersensitive response (HR)-like cell death and or leaf maceration. A strain with a mutation in the type II secretion system caused HR-like plant cell death but no maceration. P. carotovorum was unable to impede callose deposition in N. benthamiana leaves, suggesting that P. carotovorum does not suppress this basal immunity function. Within 24 hpi, there was callose deposition along leaf veins and examination showed that the pathogen cells were localized along the veins. To further examine HR-like plant cell death induced by P. carotovorum, gene expression profiles in N. benthamiana leaves inoculated with wild-type and mutant P. carotovorum and Pseudomonas syringae strains were compared. The N. benthamiana gene expression profile of leaves infiltrated with Pectobacterium carotovorum was similar to leaves infiltrated with a Pseudomonas syringae T3SS mutant. These data support a model where Pectobacterium carotovorum uses the T3SS to induce plant cell death in order to promote leaf maceration rather than to suppress plant immunity.     

The HrpN effector of Erwinia amylovora, which is involved in type III translocation, contributes directly or indirectly to callose elicitation on apple leaves

Erwinia amylovora is responsible for fire blight of apple and pear trees. Its pathogenicity depends on a type III secretion system (T3SS) mediating the translocation of effectors into the plant cell. The DspA/E effector suppresses callose deposition on apple leaves. We found that E. amylovora and Pseudomonas syringae DC3000 tts mutants or peptide flg22 do not trigger callose deposition as strongly as the dspA/E mutant on apple leaves. This suggests that, on apple leaves, callose deposition is poorly elicited by pathogen-associated molecular patterns (PAMPs) such as flg22 or other PAMPs harbored by tts mutants and is mainly elicited by injected effectors or by the T3SS itself. Callose elicitation partly depends on HrpW because an hrpW-dspA/E mutant elicits lower callose deposition than a dspA/E mutant. Furthermore, an hrpN-dspA/E mutant does not trigger callose deposition, indicating that HrpN is required to trigger this plant defense reaction. We showed that HrpN plays a general role in the translocation process. Thus, the HrpN requirement for callose deposition may be explained by its role in translocation: HrpN could be involved in the translocation of other effectors inducing callose deposition. Furthermore, HrpN may also directly contribute to the elicitation process because we showed that purified HrpN induces callose deposition.     

Hydrogen peroxide production in wheat leaves infected with the fungus Septoria nodorum Berk. Strains with different virulence

The effect of two strains of the phytopathogenic fungus Septoria nodorum Berk. of different virulence on the intensity of local generation of hydrogen peroxide in common wheat leaves and the role of oxidoreductases in this process was studied. Differences in the pattern of hydrogen peroxide production in wheat plants infected with high- and low-virulence pathogen strains have been found. The low-virulent S. nodorum strain caused a long-term hydrogen peroxide (H₂O₂) generation in the infection zone, whereas the inoculation of leaves with the highly virulent strain resulted in a transient short-term increase in the H₂O₂ concentration at the initial moment of contact between the plant and the fungus. It was shown that the low level of H₂O₂ production by plant cells at the initial stages of pathogenesis facilitates S. nodorum growth and development. The decrease in the H₂O₂ concentration induced by the highly virulent S. nodorum strain is determined by inhibition of the oxalate oxidase activity in plant tissues and by the ability of the fungus to actively synthesize an extracellular catalase. The pattern of hydrogen peroxide generation at the initial stages of septoriosis may serve as an index of virulence of S. nodorum population.     

AvrAC(Xcc8004), a type III effector with a leucine-rich repeat domain from Xanthomonas campestris pathovar campestris confers avirulence in vascular tissues of Arabidopsis thaliana ecotype Col-0

Xanthomonas campestris pathovar campestris causes black rot, a vascular disease on cruciferous plants, including Arabidopsis thaliana. The gene XC1553 from X. campestris pv. campestris strain 8004 encodes a protein containing leucine-rich repeats (LRRs) and appears to be restricted to strains of X. campestris pv. campestris. LRRs are found in a number of type III-secreted effectors in plant and animal pathogens. These prompted us to investigate the role of the XC1553 gene in the interaction between X. campestris pv. campestris and A. thaliana. Translocation assays using the hypersensitive-reaction-inducing domain of X. campestris pv. campestris AvrBs1 as a reporter revealed that XC1553 is a type III effector. Infiltration of Arabidopsis leaf mesophyll with bacterial suspensions showed no differences between the wild-type strain and an XC1553 gene mutant; both strains induced disease symptoms on Kashmir and Col-0 ecotypes. However, a clear difference was observed when bacteria were introduced into the vascular system by piercing the central vein of leaves. In this case, the wild-type strain 8004 caused disease on the Kashmir ecotype, but not on ecotype Col-0; the XC1553 gene mutant became virulent on the Col-0 ecotype and still induced disease on the Kashmir ecotype. Altogether, these data show that the XC1553 gene, which was renamed avrAC_Xcc8004, functions as an avirulence gene whose product seems to be recognized in vascular tissues.     

A chromosomally encoded type III secretion pathway in Yersinia enterocolitica is important in virulence

Numerous Gram-negative bacteria use a type III, or contact dependent, secretion system to deliver proteins into the cytosol of host cells. All of these systems identified to date have been shown to have a role in pathogenesis. We have identified 13 genes on the Yersinia enterocolitica chromosome that encode a type III secretion apparatus plus two associated putative regulatory genes. In order to determine the function of this chromosomally-encoded secretion apparatus, we created an in frame deletion of a gene that has homology to the hypothesized inner membrane pore, ysaV. The ysaV mutant strain failed to secrete eight proteins, called Ysps, normally secreted by the parental strain when grown at 28 degrees C in Luria-Bertani (LB) broth supplemented with 0.4 M NaCl. Disruption of the ysaV gene had no effect on motility or phospholipase activity, suggesting this chromosomally encoded type III secretion pathway is distinct from the flagella secretion pathway of Y. enterocolitica. Deletion of the ysaV gene in a virulence plasmid positive strain had no effect on in vitro secretion of Yops by the plasmid-encoded type III secretion apparatus. Secretion of the Ysps was unaffected by the presence or absence of the virulence plasmid, suggesting the chromosomally encoded and plasmid-encoded type III secretion pathways act independently. Y. enterocolitica thus has three type III secretion pathways that appear to act independently. The ysaV mutant strain was somewhat attenuated in virulence compared with the wild type in the mouse oral model of infection (an approximately 0.9 log difference in LD50). The ysaV mutant strain was nearly as virulent as the wild type when inoculated intraperitoneally in the mouse model. A ysaV probe hybridized to sequences in other Yersinia spp. and homologues were found in the incomplete Y. pestis genome sequence, indicating a possible role for this system throughout the genus.     

Mutation of the Erwinia amylovora argD Gene Causes Arginine Auxotrophy,Nonpathogenicity in Apples,and Reduced Virulence in Pears

Fire blight is caused by Erwinia amylovora and is the most destructive bacterial disease of apples and pears worldwide. In this study, we found that E. amylovora argD(1000)::Tn5, an argD Tn5 transposon mutant that has the Tn5 transposon inserted after nucleotide 999 in the argD gene-coding region, was an arginine auxotroph that did not cause fire blight in apple and had reduced virulence in immature pear fruits. The E. amylovora argD gene encodes a predicted N-acetylornithine aminotransferase enzyme, which is involved in the production of the amino acid arginine. A plasmid-borne copy of the wild-type argD gene complemented both the nonpathogenic and the arginine auxotrophic phenotypes of the argD(1000)::Tn5 mutant. However, even when mixed with virulent E. amylovora cells and inoculated onto immature apple fruit, the argD(1000)::Tn5 mutant still failed to grow, while the virulent strain grew and caused disease. Furthermore, the pCR2.1-argD complementation plasmid was stably maintained in the argD(1000)::Tn5 mutant growing in host tissues without any antibiotic selection. Therefore, the pCR2.1-argD complementation plasmid could be useful for the expression of genes, markers, and reporters in E. amylovora growing in planta, without concern about losing the plasmid over time. The ArgD protein cannot be considered an E. amylovora virulence factor because the argD(1000)::Tn5 mutant was auxotrophic and had a primary metabolism defect. Nevertheless, these results are informative about the parasitic nature of the fire blight disease interaction, since they indicate that E. amylovora cannot obtain sufficient arginine from apple and pear fruit tissues or from apple vegetative tissues, either at the beginning of the infection process or after the infection has progressed to an advanced state.     

FliC,a Flagellin Protein,Is Essential for the Growth and Virulence of Fish Pathogen Edwardsiella tarda

Edwardsiella tarda is a flagellated Gram-negative bacterium which causes edwardsiellosis in fish. FliC, as a flagellar filament structural protein, is hypothesized to be involved in the pathogenesis of infection. In this study, a fliC in-frame deletion mutant of a virulent isolate of E. tarda was constructed through double crossover allelic exchange by means of the suicide vector pRE112, and its virulence-associated phenotypes and pathogenicity were tested. It was found that the deletion of fliC significantly decreased the diameter of flagella filaments. In addition, the mutant showed reduced pathogenicity to fish by increasing the LD₅₀ value for 100-fold compared to the wild-type strain, as well as showed impaired bacterial growth, reduced motility, decreased biofilm formation and reduced levels of virulence-associated protein secretion involved in the type III secretion system (TTSS). The phenotypic characteristics of the fliC deletion mutant uncovered in this investigation suggest that fliC plays an essential role in normal flagellum function, bacterial growth, protein secretion by TTSS and bacterial virulence.     

A Phenotypic,Molecular and Biochemical Characterization of the First Cisgenic Scab-Resistant Apple Variety ‘Gala’

Scab resistance is one of the most important goals of apple breeding, typically achieved by time-consuming and expensive conventional breeding techniques. Cisgenesis, which is the genetic modification of a recipient organism with genes from a crossable—sexually compatible—organism, is a promising tool for plant breeding to develop disease resistance in a rapid way. A cisgenic, scab-resistant line of the apple variety ‘Gala’ expressing the native apple scab resistance gene Rvi6 (formerly HcrVf2) under control of its own regulatory sequences has been recently developed. In this paper, we present the results from a phenotypic, molecular and biochemical evaluation of clonal replicates of this line (C11.1.53). The phenotype (shoot length, shoot diameter, internode length, number of leaves, leaf length and leaf width) of C11.1.53 was compared to that of the Gala parental background over a period of 108 days. Only a few statistically significant differences were detected, which are probably due to small differences in the quality of the budwood used for grafting rather than effects related to the presence of the cisgene. As the expression of a resistance gene can affect the downstream cascade of plant defence responses, a selection of apple defence-related genes was analyzed by quantitative real-time PCR analysis. These genes are also known as major allergen genes in apple. Even if three out of ten apple allergen genes tested in the leaves differed in the cisgenic line compared to both Gala (background) and ‘Florina’ (the variety from which the Rvi6 gene was cloned), using 2D-PAGE, we were unable to find any significant difference in the expressed proteomes of the leaves of C11.1.53 compared to Gala. Results are discussed in the context of a possible use of cisgenic lines for fruit crop improvement.     

The Role of relA and spoT in Yersinia pestis KIM5+ Pathogenicity

The ppGpp molecule is part of a highly conserved regulatory system for mediating the growth response to various environmental conditions. This mechanism may represent a common strategy whereby pathogens such as Yersinia pestis, the causative agent of plague, regulate the virulence gene programs required for invasion, survival and persistence within host cells to match the capacity for growth. The products of the relA and spoT genes carry out ppGpp synthesis. To investigate the role of ppGpp on growth, protein synthesis, gene expression and virulence, we constructed a ΔrelA ΔspoT Y. pestis mutant. The mutant was no longer able to synthesize ppGpp in response to amino acid or carbon starvation, as expected. We also found that it exhibited several novel phenotypes, including a reduced growth rate and autoaggregation at 26°C. In addition, there was a reduction in the level of secretion of key virulence proteins and the mutant was>1,000-fold less virulent than its wild-type parent strain. Mice vaccinated subcutaneously (s.c.) with 2.5×10⁴ CFU of the ΔrelA ΔspoT mutant developed high anti-Y. pestis serum IgG titers, were completely protected against s.c. challenge with 1.5×10⁵ CFU of virulent Y. pestis and partially protected (60% survival) against pulmonary challenge with 2.0×10⁴ CFU of virulent Y. pestis. Our results indicate that ppGpp represents an important virulence determinant in Y. pestis and the ΔrelA ΔspoT mutant strain is a promising vaccine candidate to provide protection against plague.     

Plasmid Content and Tumor Initiation Complementation by Agrobacterium tumefaciens IIBNV6

Avirulent strains IIBNV6 and NT1, derived from virulent strains of Agrobacterium tumefaciens, were tested for their ability to enhance tumor initiation (complement) on coinoculation with tumorigenic strains. Strain NT1, cured of the Agrobacterium virulence plasmid, failed to complement when inoculated with its virulent parental strain or with other virulent strains. Strain IIBNV6, however, complemented with all virulent strains tested. Attachment to host wound sites by both strain IIBNV6 and the virulent strain was essential for this effect. Inoculation of the tumorigenic strain at different times on leaves previously inoculated with IIBNV6 showed that the capacity to complement is lost during the period between 4 and 8 h after IIBNV6 inoculation. The rate of tumor appearance obtained with an inoculum containing IIBNV6 and a virulent auxotrophic strain was characteristic of the appearance rate obtained with prototrophic bacteria. Evidence is summarized which suggests that strain IIBNV6 can induce tumors when supplied with a substance produced or induced by a virulent bacterium at a separate site. A deoxyribonucleic acid plasmid about 40% the size of the Agrobacterium virulence plasmid was obtained from strain IIBNV6. We propose that this plasmid accounts for the ability of strain IIBNV6 to complement and that it contains part of the genetic information necessary for tumor initiation.     

AmyR Is a Novel Negative Regulator of Amylovoran Production in Erwinia amylovora

In this study, we attempted to understand the role of an orphan gene amyR in Erwinia amylovora, a functionally conserved ortholog of ybjN in Escherichia coli, which has recently been characterized. Amylovoran, a high molecular weight acidic heteropolymer exopolysaccharide, is a virulent factor of E. amylovora. As reported earlier, amylovoran production in an amyR knockout mutant was about eight-fold higher than that in the wild type (WT) strain of E. amylovora. When a multicopy plasmid containing the amyR gene was introduced into the amyR mutant or WT strains, amylovoran production was strongly inhibited. Furthermore, amylovoran production was also suppressed in various amylovoran-over-producing mutants, such as grrSA containing multicopies of the amyR gene. Consistent with amylovoran production, an inverse correlation was observed between in vitro expression of amyR and that of amylovoran biosynthetic genes. However, both the amyR knockout mutant and over-expression strains showed reduced levan production, another exopolysaccharide produced by E. amylovora. Virulence assays demonstrated that while the amyR mutant was capable of inducing slightly greater disease severity than that of the WT strain, strains over-expressing the amyR gene did not incite disease on apple shoots or leaves, and only caused reduced disease on immature pear fruits. Microarray studies revealed that amylovoran biosynthesis and related membrane protein-encoding genes were highly expressed in the amyR mutant, but down-regulated in the amyR over-expression strains in vitro. Down-regulation of amylovoran biosynthesis genes in the amyR over-expression strain partially explained why over-expression of amyR led to non-pathogenic or reduced virulence in vivo. These results suggest that AmyR plays an important role in regulating exopolysaccharide production, and thus virulence in E. amylovora.     

Response to Xanthomonas campestris pv. vesicatoria in tomato involves regulation of ethylene receptor gene expression

Although ethylene regulates a wide range of defense-related genes, its role in plant defense varies greatly among different plant-microbe interactions. We compared ethylene's role in plant response to virulent and avirulent strains of Xanthomonas campestris pv. vesicatoria in tomato (Lycopersicon esculentum Mill.). The ethylene-insensitive Never ripe (Nr) mutant displays increased tolerance to the virulent strain, while maintaining resistance to the avirulent strain. Expression of the ethylene receptor genes NR and LeETR4 was induced by infection with both virulent and avirulent strains; however, the induction of LeETR4 expression by the avirulent strain was blocked in the Nr mutant. To determine whether ethylene receptor levels affect symptom development, transgenic plants overexpressing a wild-type NR cDNA were infected with virulent X. campestris pv. vesicatoria. Like the Nr mutant, the NR overexpressors displayed greatly reduced necrosis in response to this pathogen. NR overexpression also reduced ethylene sensitivity in seedlings and mature plants, indicating that, like LeETR4, this receptor is a negative regulator of ethylene response. Therefore, pathogen-induced increases in ethylene receptors may limit the spread of necrosis by reducing ethylene sensitivity.     

Involvement of fengycin-type lipopeptides in the multifaceted biocontrol potential of Bacillus subtilis

In this work, the potential of Bacillus subtilis strain M4 at protecting plants against fungal diseases was demonstrated in different pathosystems. We provide evidence for the role of secreted lipopeptides, and more particularly of fengycins, in the protective effect afforded by the strain against damping-off of bean seedlings caused by Pythium ultimum and against gray mold of apple in post-harvest disease. This role was demonstrated by the strong biocontrol activity of lipopeptide-enriched extracts and through the detection of inhibitory quantities of fengycins in infected tissues. Beside such a direct antagonism of the pathogen, we show that root pre-inoculation with M4 enabled the host plant to react more efficiently to subsequent pathogen infection on leaves. Fengycins could also be involved in this systemic resistance-eliciting effect of strain M4, as these molecules may induce the synthesis of plant phenolics involved in or derived from the defense-related phenylpropanoid metabolism. Much remains to be discovered about the mechanisms by which Bacillus spp suppress disease. Through this study on strain M4, we reinforce the interest in B. subtilis as a pathogen antagonist and plant defense-inducing agent. The secretion of cyclic fengycin-type lipopeptides may be tightly related to the expression of these two biocontrol traits.     

Oxalic acid, a pathogenicity factor for Sclerotinia sclerotiorum, suppresses the oxidative burst of the host plant

Effective pathogenesis by the fungus Sclerotinia sclerotiorum requires the secretion of oxalic acid. Studies were conducted to determine whether oxalate aids pathogen compatibility by modulating the oxidative burst of the host plant. Inoculation of tobacco leaves with an oxalate-deficient nonpathogenic mutant of S. sclerotiorum induced measurable oxidant biosynthesis, but inoculation with an oxalate-secreting strain did not. Oxalate inhibited production of H(2)O(2) in tobacco and soybean cultured cell lines with a median inhibitory concentration of approximately 4 to 5 mM, a concentration less than that measured in preparations of the virulent fungus. Several observations also indicate that the inhibitory effects of oxalate are largely independent of both its acidity and its affinity for Ca(2)+. These and other data demonstrate that oxalate may inhibit a signaling step positioned upstream of oxidase assembly/activation but downstream of Ca(2)+ fluxes into the plant cell cytosol.