Emilia López-Solanilla

Inactivation of the sapA to sapF locus of Erwinia chrysanthemi reveals common features in plant and animal bacterial pathogenesis.

We investigated the role in pathogenesis of bacterial resistance to plant antimicrobial peptides. The sapA to sapF (for sensitive to antimicrobial peptides) operon from the pathogenic bacterium Erwinia chrysanthemi has been characterized. It has five open reading frames that are closely related (71% overall amino acid identity) and are in the same order as those of the sapA to sapF operon from Salmonella typhimurium. An E. chrysanthemi sap mutant strain was constructed by marker exchange. This mutant was more sensitive than was the wild type to wheat α-thionin and to snakin-1, which is the most abundant antimicrobial peptide from potato tubers. This mutant was also less virulent than was the wild-type strain in potato tubers: lesion area was 37% that of the control, and growth rate was two orders of magnitude lower. These results indicate that the interaction of antimicrobial peptides from the host with the sapA to sapF operon from the pathogen plays a similar role in animal and in plant bacterial pathogenesis.

Antibiotic activities of peptides, hydrogen peroxide and peroxynitrite in plant defence.

Genes encoding plant antibiotic peptides show expression patterns that are consistent with a defence role. Transgenic over‐expression of defence peptide genes is potentially useful to engineer resistance of plants to relevant pathogens. Pathogen mutants that are sensitive to plant peptides in vitro have been obtained and a decrease of their virulence in planta has been observed, which is consistent with their hypothetical defence role. A similar approach has been followed to elucidate the potential direct anti‐microbial role of hydrogen peroxide. Additionally, a scavenger of peroxynitrite has been used to investigate its involvement in plant defence.

A bacterial cysteine protease effector protein interferes with photosynthesis to suppress plant innate immune responses

The bacterial pathogen Pseudomonas syringae pv tomato DC3000 suppresses plant innate immunity with effector proteins injected by a type III secretion system (T3SS). The cysteine protease effector HopN1, which reduces the ability of DC3000 to elicit programmed cell death in non‐host tobacco, was found to also suppress the production of defence‐associated reactive oxygen species (ROS) and callose when delivered by Pseudomonas fluorescens heterologously expressing a P. syringae T3SS. Purified His6‐tagged HopN1 was used to identify tomato PsbQ, a member of the oxygen evolving complex of photosystem II (PSII), as an interacting protein. HopN1 localized to chloroplasts and both degraded PsbQ and inhibited PSII activity in chloroplast preparations, whereas a HopN1D299A non‐catalytic mutant lost these abilities. Gene silencing of NtPsbQ in tobacco compromised ROS production and programmed cell death by DC3000. Our data reveal PsbQ as a contributor to plant immunity responses and a target for pathogen suppression.

Antifungal Activity of a Plant Cystatin

Purified chestnut cystatin inhibited the growth of the phytopathogenic fungi Botrytis cinerea, Colletotrichum graminicola, and Septoria nodorum, but not that of the saprophyte Trichoderma viride. Furthermore, the cystatin strongly affected the protease activity of B. cinerea but had no effect on the protease activity of T. viride. These results suggest that chestnut cystatin contributes to plant defense against phytopathogenic fungi.

Inhibition of plant-pathogenic fungi by the barley cystatin Hv-CPI (gene Icy) is not associated with its cysteine-proteinase inhibitory properties.

The recombinant barley cystatin Hv-CPI inhibited the growth of three phytopathogenic fungi (Botrytis cinerea, Colletotrichum graminicola, and Plectosphaerella cucumerina) and the saprotrophic fungus Trichoderma viride. Several mutants of barley cystatin were generated by polymerase chain reaction approaches and both their antifungal and their cysteine-proteinase inhibitory properties investigated. Point mutants R38-->G, Q63-->L, and Q63-->P diminished their capacity for inhibiting papain and cathepsin B, retaining their antifungal properties. However, mutant C68-->G was more active for papain and cathepsin B than the wild type. These results indicate that in addition to the consensus cystatin-reactive site, Q63-V64-V65-A66-G67, the A37-R38-F39-A40-V41 region, common to all cereal cystatins, and the C68 residue are important for barley cystatin activity. On the other hand, the K92-->P mutant is inactive as a fungicide, but still retains measurable inhibitory activity for papain and cathepsin B. Against B. cinerea, the antifungal effect of Hv-CPI and of its derived mutants does not always correlate with their activities as proteinase inhibitors, because the Q63-->P mutant is inactive as a cystatin, while still inhibiting fungal growth, and the K92-->P mutant shows the reciprocal effects. These data indicate that inhibition of plant-pathogenic fungi by barley cystatin is not associated with its cysteine-proteinase inhibitory activity. Moreover, these results are corroborated by the absence of inhibition of intra- and extramycelia-proteinase activities by barley cystatin and by other well-known inhibitors of cysteine-proteinase activity in the fungal zymograms of B. cinerea.

Role of motility and chemotaxis in the pathogenesis of Dickeya dadantii 3937 (ex Erwinia chrysanthemi 3937)

Dickeya dadantii 3937 (ex Erwinia chrysanthemi), a member of the Enterobacteriaceae, causes soft rot in many economically important crops. A successful pathogen has to reach the interior of the plant in order to cause disease. To study the role of motility and chemotaxis in the pathogenicity of D. dadantii 3937, genes involved in the chemotactic signal transduction system (cheW, cheB, cheY and cheZ) and in the structure of the flagellar motor (motA) were mutagenized. All the mutant strains grew like the wild-type in culture media, and the production and secretion of pectolytic enzymes was not affected. As expected, the swimming ability of the mutant strains was reduced with respect to the wild-type: motA (94%), cheY (80%), cheW (74%), cheB (54%) and cheZ (48%). The virulence of the mutant strains was analysed in chicory, Saintpaulia and potato. The mutant strains were also tested for their capability to enter into Arabidopsis leaves. All the mutants showed a significant decrease of virulence in certain hosts; however, the degree of virulence reduction varied depending on the virulence assay. The ability to penetrate Arabidopsis leaves was impaired in all the mutants, whereas the capacity to colonize potato tubers after artificial inoculation was affected in only two mutant strains. In general, the virulence of the mutants could be ranked as motA<cheY<cheB=cheW<cheZ, which correlated with the degree to which swimming was affected. These results clearly indicate that motility plays an important role in the pathogenicity of this bacterium.

The Erwinia chrysanthemi phoP-phoQ operon plays an important role in growth at low pH, virulence and bacterial survival in plant tissue.

We have studied the role of acidic pH as a barrier for the colonization of the plant apoplast by Erwinia chrysanthemi. A minitransposon containing a promoterless reporter gene, gus, was used for random mutagenesis of the bacterial genome. An acid‐sensitive mutant, named BT119, was isolated and had the following differential features with respect to the wild‐type strain: (i) inability to grow at pH ≤ 5.5; (ii) decreased survival at acid pH and in plant tissues; (iii) increased susceptibility to antimicrobial peptides; (iv) decreased virulence in chicory leaves and pear fruits; (v) reduced polygalacturonase production; and (vi) reduced ability to alkalinize chicory tissues after infection. The sequence of the interrupted gene was highly similar to the phoQ gene, which is involved in environmental sensing in several bacteria, such as Yersinia pseudotuberculosis, Erwinia carotovora, Salmonella typhimurium and Escherichia coli and thus, this designation was used for the E. chrysanthemi system. This gene was induced at low Mg2+ concentrations and in planta. These results suggest that E. chrysanthemi PhoP‐PhoQ system plays an important role in bacterial survival in plant tissues during the initial infection stages.

Relative Effects on Virulence of Mutations in the sap, pel, and hrp Loci of Erwinia chrysanthemi

We constructed strains of Erwinia chrysanthemi EC16 with multiple mutations involving three virulence systems in this bacterium, namely pel (coding for the major pectate lyases pelABCE), hrp (hypersensitive response and pathogenicity), and sap (sensitivity to antimicrobial peptides). The relative effects on virulence of those mutations have been analyzed on potato tubers and chicory leaves. In potato tubers, the sap mutation (BT105) had a greater effect in the reduction of the virulence than the pel (CUCPB5006) and hrp (CUCPB5039) mutations. This reduction was similar to that observed in the pel-hrp double mutant (CUCPB5037). The analysis of the strains affected in Pel-Sap (BT106), Hrp-Sap (BT107), and Pel-Hrp-Sap (BT108) suggested that the effects of these mutations are additive. In chicory leaves, the mutation in the sap locus appeared to have a greater effect than in potato tubers. The competitive indices of strains BT105, UM1005 (Pel-), CUCPB5039, and CUCPB5037 have been estimated in vivo and in vitro. These results indicate that the mutation in the hrp locus can be complemented in vivo by coinfection, whereas the mutations in pel and sap cannot.

Evidence Against a Direct Antimicrobial Role of H2O2 in the Infection of Plants by Erwinia chrysanthemi

We have investigated the role of bacterial resistance to oxidative stress in pathogenesis. The oxyR gene from the pathogenic bacterium Erwinia chrysanthemi has been characterized. It is closely related to that found in Escherichia coli (88% overall amino acid identity). An E. chrysanthemi oxyR mutant strain was constructed by marker exchange. After induction with a sublethal dose of H2O2, this mutant was more sensitive to H2O2 and showed reduced levels of catalase and glutathione reductase activities, compared with the wild type. The oxyR mutant was unable to form individual colonies on agar plates unless catalase was added exogenously. However, it retained full virulence in potato tubers and tobacco leaves. These results suggest that the host-produced H2O2 has no direct antimicrobial effect on the interaction of E. chrysanthemi with the two plant species.

The Role of Several Multidrug Resistance Systems in Erwinia chrysanthemi Pathogenesis

The role of several multidrug resistance (MDR) systems in the pathogenicity of Erwinia chrysanthemi 3937 was analyzed. Using the blast algorithm, we have identified several MDR systems in the E. chrysanthemi genome and selected two acridine resistance (Acr)-like systems, two Emr-like systems, and one member of the major facilitator super-family family to characterize. We generated mutants in genes encoding for these systems and analyzed the virulence of the mutant strains in different hosts and their susceptibility to antibiotics, detergents, dyes, and plant compounds. We have observed that the mutant strains are differentially affected in their virulence in different hosts and that the susceptibility to toxic substances is also differential. Both Acr systems seem to be implicated in the resistance to the plant antimicrobial peptide thionin. Similarly, the emr1AB mutant is unable to grow in the presence of the potato protein tuber extract and shows a decreased virulence in this tissue. These results indicate that the function of these systems in plants could be related to the specificity to extrude a toxic compound that is present in a given host.