The bacterial effector DspA/E is toxic in Arabidopsis thaliana and is required for multiplication and survival of fire blight pathogen.

The type III effector DspA/E is an essential pathogenicity factor of the phytopathogenic bacterium Erwinia amylovora. We showed that DspA/E was required for transient bacterial growth in nonhost Arabidopsis thaliana leaves, as an E. amylovora dspA/E mutant was unable to grow. We expressed DspA/E in A. thaliana transgenic plants under the control of an oestradiol-inducible promoter, and found that DspA/E expressed in planta restored the growth of a dspA/E mutant. DspA/E expression in these transgenic plants led to the modulation by at least two-fold of the expression of 384 genes, mostly induced (324 genes). Both induced and repressed genes contained high proportions of defence genes. DspA/E expression ultimately resulted in plant cell death without requiring a functional salicylic acid signalling pathway. Analysis of A. thaliana transgenic seedlings expressing a green fluorescent protein (GFP):DspA/E fusion indicated that the fusion protein could only be detected in a few cells per seedling, suggesting the degradation or absence of accumulation of DspA/E in plant cells. Consistently, we found that DspA/E repressed plant protein synthesis when injected by E. amylovora or when expressed in transgenic plants. Thus, we conclude that DspA/E is toxic to A. thaliana: it promotes modifications, among which the repression of protein synthesis could be determinant in the facilitation of necrosis and bacterial growth.

EDS1 contributes to nonhost resistance of Arabidopsis thaliana against Erwinia amylovora.

Erwinia amylovora causes fire blight in rosaceous plants. In nonhost Arabidopsis thaliana, E. amylovora triggers necrotic symptoms associated with transient bacterial multiplication, suggesting either that A. thaliana lacks a susceptibility factor or that it actively restricts E. amylovora growth. Inhibiting plant protein synthesis at the time of infection led to an increase in necrosis and bacterial multiplication and reduced callose deposition, indicating that A. thaliana requires active protein synthesis to restrict E. amylovora growth. Analysis of the callose synthase-deficient pmr4-1 mutant indicated that lack of callose deposition alone did not lead to increased sensitivity to E. amylovora. Transcriptome analysis revealed that approximately 20% of the genes induced following E. amylovora infection are related to defense and signaling. Analysis of mutants affected in NDR1 and EDS1, two main components of the defense-gene activation observed, revealed that E. amylovora multiplied ten times more in the eds1-2 mutant than in the wild type but not in the ndr1-1 mutant. Analysis of mutants affected in three WRKY transcription factors showing EDS1-dependent activation identified WRKY46 and WRKY54 as positive regulators and WRKY70 as a negative regulator of defense against E. amylovora. Altogether, we show that EDS1 is a positive regulator of nonhost resistance against E. amylovora in A. thaliana and hypothesize that it controls the production of several effective defenses against E. amylovora through the action of WRKY46 and WRKY54, while WRKY70 acts as a negative regulator.

Characterization of a new, nonpathogenic mutant of Botrytis cinerea with impaired plant colonization capacity.

Botrytis cinerea is a necrotrophic pathogen that attacks more than 200 plant species. Here, the nonpathogenic mutant A336, obtained via insertional mutagenesis, was characterized. Mutant A336 was nonpathogenic on leaves and fruits, on intact and wounded tissue, while still able to penetrate the host plant. It grew normally in vitro on rich media but its conidiation pattern was altered. The mutant did not produce oxalic acid and exhibited a modified regulation of the production of some secreted proteins (acid protease 1 and endopolygalacturonase 1). Culture filtrates of the mutant triggered an important oxidative burst in grapevine (Vitis vinifera) suspension cells, and the mutant-plant interaction resulted in the formation of hypersensitive response-like necrosis. Genetic segregation analyses revealed that the pathogenicity phenotype was linked to a single locus, but showed that the mutated gene was not tagged by the plasmid pAN7-1. Mutant A336 is the first oxalate-deficient mutant to be described in B. cinerea and it differs from all the nonpathogenic B. cinerea mutants described to date.

PecS and PecT coregulate the synthesis of HrpN and pectate lyases, two virulence determinants in Erwinia chrysanthemi 3937.

Erwinia chrysanthemi strain 3937 is a necrotrophic bacterial plant pathogen. Pectinolytic enzymes and, in particular, pectate lyases play a key role in soft rot symptoms; however, the efficient colonization of plants by E. chrysanthemi requires additional factors. These factors include HrpN (harpin), a heat-stable, glycine-rich hydrophilic protein, which is secreted by the type III secretion system. We investigated the expression of hrpN in E. chrysanthemi 3937 in various environmental conditions and different regulatory backgrounds. Using lacZ fusions, hrpN expression was markedly influenced by the carbon source, osmolarity, growth phase, and growth substrate. hrpN was repressed when pectinolysis started and negatively regulated by the repressors of pectate lyase synthesis, PecS and PecT. Primer extension data and in vitro DNA-protein interaction experiments support a model whereby PecS represses hrpN expression by binding to the hrpN regulatory region and inhibiting transcript elongation. The results suggest coordinated regulation of HrpN and pectate lyases by PecS and PecT. A putative model of the synthesis of these two virulence factors in E. chrysanthemi during pathogenesis is presented.

A CFTR chloride channel activator prevents HrpN(ea)-induced cell death in Arabidopsis thaliana suspension cells.

Erwinia amylovora is a necrogenic bacterium that causes fire blight of the Maloideae subfamily of Roseacae, such as apple and pear. It provokes necrosis in aerial parts of susceptible host plants and the typical hypersensitive reaction in non-host plants. The secreted harpin, HrpN ea, is able by itself to induce an active cell death in non-host plants. Ion flux modulations were shown to be involved early in such processes but very few data are available on the plasma membrane ion channel activities responsible for the pathogen-induced ion fluxes. We show here that HrpN ea induces cell death in non-host Arabidopsis thaliana suspension cells. We further show that two cystic fibrosis transmembrane conductance regulator modulators, glibenclamide and bromotetramisole, can regulate anion channel activities and HrpN ea-induced cell death.

hrp genes of Erwinia chrysanthemi 3937 are important virulence factors.

We developed improved virulence assays for Erwinia chrysanthemi 3937 on African violet varieties and devised a new method for the construction of precise bacterial gene knockouts. These methods were tested by constructing mutations in genes suspected to be involved with plant interactions. The virulence of the hrpG and hrcC mutant strains (both gene products presumed to be involved in protein secretion) was greatly reduced on leaves of semitolerant African violet varieties. An hrpN mutant strain produced delayed symptoms on African violet leaves and an hrpN delta pel (delta pel = five major pectate lyase genes deleted) double mutant was nonpathogenic. The hrcC and hrpG mutants did not produce a rapid hypersensitive response (HR) in tobacco, unlike the wild-type bacterium, and the hrpN mutant gave a reduced HR. The results, therefore, establish the importance of hrp genes in the virulence of E. chrysanthemi and their ability to elicit HR on nonhosts. The data also suggest that other effector proteins secreted by the Hrp system are required for full virulence and HR elicitation.