Potato StMPK7 is a downstream component of StMKK1 and promotes resistance to the oomycete pathogen Phytophthora infestans

A cascade formed by phosphorylation events of mitogen-activated protein kinases (MAPKs) takes part in plant stress responses. However, the roles of these MAPKs in resistance of potato (Solanum tuberosum) against Phytophthora pathogens is not well studied. Our previous work showed that a Phytophthora infestans RXLR effector targets and stabilizes the negative regulator of MAPK kinase 1 of potato (StMKK1). Because in Arabidopsis thaliana the AtMPK4 is the downstream phosphorylation target of AtMKK1, we performed a phylogenetic analysis and found that potato StMPK4/6/7 are closely related and are orthologs of AtMPK4/5/11/12. Overexpression of StMPK4/7 enhances plant resistance to P. infestans and P. parasitica. Yeast two-hybrid analysis revealed that StMPK7 interacts with StMKK1, and StMPK7 is phosphorylated on flg22 treatment and by expressing constitutively active StMKK1 (CA-StMKK1), indicating that StMPK7 is a direct downstream signalling partner of StMKK1. Overexpression of StMPK7 in potato enhances potato resistance to P. infestans. Constitutively active StMPK7 (CA-StMPK7; StMPK7^{D198G, E202A}) was found to promote immunity to Phytophthora pathogens and to trigger host cell death when overexpressed in Nicotiana benthamiana leaves. Cell death triggered by CA-StMPK7 is SGT1/RAR1-dependent. Furthermore, cell death triggered by CA-StMPK7 is suppressed on coexpression with the salicylate hydroxylase NahG, and StMPK7 activation promotes salicylic acid (SA)-responsive gene expression. We conclude that potato StMPK7 is a downstream signalling component of the phosphorelay cascade involving StMKK1 and StMPK7 plays a role in immunity to Phytophthora pathogens via an SA-dependent signalling pathway.     

Alteration of pathogenicity-linked life-history traits by resistance of its host Solanum tuberosum impacts sexual reproduction of the plant pathogenic oomycete Phytophthora infestans

Although sexual reproduction implies a cost, it represents an evolutionary advantage for the adaptation and survival of facultative sexual pathogens. Understanding the maintenance of sex in pathogens requires to analyse how host resistance will impact their sexual reproduction through the alteration of their life-history traits. We explored this experimentally using potato (Solanum tuberosum) and one of its pathogens, the heterothallic oomycete Phytophthora infestans. Sexual reproduction was highest on hosts favouring asexual multiplication of the pathogen, suggesting similar nutritional requirements for both sexual and asexual sporulation. Sexual reproduction was also highest on hosts decreasing the latent period, probably because of a trade-off between growth and reproduction. Distinguishing host effects on each pathogenic trait remains however uneasy, as most life-history traits linked to pathogenicity were not independent of each other. We argue that sexual reproduction of P. infestans is an adaptation to survive when the host is susceptible and rapidly destroyed.     

Mycobacterial proteomics: analysis of expressed proteomes and post-translational modifications to identify candidate virulence factors

The Mycobacterium tuberculosis bacillus has a number of unique features that make it a particularly effective human pathogen. Although genomic analysis has added to our current understanding of the molecular basis by which M. tuberculosis damages its host, proteomics may be better suited to describe the dynamic interactions between mycobacterial and host systems that underpin this disease. The M. tuberculosis proteome has been investigated using proteomics for over a decade, with increasingly sophisticated mass spectrometry technology and sensitive methods for comparative proteomic profiling. Deeper coverage of the M. tuberculosis proteome has led to the identification of hundreds of putative virulence determinants, as well as an unsurpassed coverage of post-translational modifications. Proteomics is therefore uniquely poised to contribute to our understanding of this pathogen, which may ultimately lead to better management of the disease.     

The C-terminal half of Phytophthora infestans RXLR effector AVR3a is sufficient to trigger R3a-mediated hypersensitivity and suppress INF1-induced cell death in Nicotiana benthamiana

The RXLR cytoplasmic effector AVR3a of Phytophthora infestans confers avirulence on potato plants carrying the R3a gene. Two alleles of Avr3a encode secreted proteins that differ in only three amino acid residues, two of which are in the mature protein. Avirulent isolates carry the Avr3a allele, which encodes AVR3aKI (containing amino acids C19, K80 and I103), whereas virulent isolates express only the virulence allele avr3a, encoding AVR3aEM (S19, E80 and M103). Only the AVR3aKI protein is recognized inside the plant cytoplasm where it triggers R3a-mediated hypersensitivity. Similar to other oomycete avirulence proteins, AVR3aKI carries a signal peptide followed by a conserved motif centered on the consensus RXLR sequence that is functionally similar to a host cell-targeting signal of malaria parasites. The interaction between Avr3a and R3a can be reconstructed by their transient co-expression in Nicotiana benthamiana. We exploited the N. benthamiana experimental system to further characterize the Avr3a-R3a interaction. R3a activation by AVR3aKI is dependent on the ubiquitin ligase-associated protein SGT1 and heat-shock protein HSP90. The AVR3aKI and AVR3aEM proteins are equally stable in planta, suggesting that the difference in R3a-mediated death cannot be attributed to AVR3aEM protein instability. AVR3aKI is able to suppress cell death induced by the elicitin INF1 of P. infestans, suggesting a possible virulence function for this protein. Structure-function experiments indicated that the 75-amino acid C-terminal half of AVR3aKI, which excludes the RXLR region, is sufficient for avirulence and suppression functions, consistent with the view that the N-terminal region of AVR3aKI and other RXLR effectors is involved in secretion and targeting but is not required for effector activity. We also found that both polymorphic amino acids, K80 and I103, of mature AVR3a contribute to the effector functions.     

Sequence diversity in the large subunit of RNA polymerase I contributes to Mefenoxam insensitivity in Phytophthora infestans

Phenylamide fungicides have been widely used for the control of oomycete‐incited plant diseases for over 30 years. Insensitivity to this chemical class of fungicide was recorded early in its usage history, but the precise protein(s) conditioning insensitivity has proven difficult to determine. To determine the genetic basis of insensitivity and to inform strategies for the cloning of the gene(s) responsible, genetic crosses were established between Mefenoxam sensitive and intermediate insensitive isolates of Phytophthora infestans, the potato late blight pathogen. F1 progeny showed the expected semi‐dominant phenotypes for Mefenoxam insensitivity and suggested the involvement of multiple loci, complicating the positional cloning of the gene(s) conditioning insensitivity to Mefenoxam. Instead, a candidate gene strategy was used, based on previous observations that the primary effect of phenylamide compounds is to inhibit ribosomal RNA synthesis. The subunits of RNA polymerase I (RNApolI) were sequenced from sensitive and insensitive isolates and F1 progeny. Single nucleotide polymorphisms (SNPs) specific to insensitive field isolates were identified in the gene encoding the large subunit of RNApolI. In a survey of field isolates, SNP T1145A (Y382F) showed an 86% association with Mefenoxam insensitivity. Isolates not showing this association belonged predominantly to one P. infestans genotype. The transfer of the ‘insensitive’ allele of RPA190 to a sensitive isolate yielded transgenic lines that were insensitive to Mefenoxam. These results demonstrate that sequence variation in RPA190 contributes to insensitivity to Mefenoxam in P. infestans.     

Composting is an effective treatment option for sanitization of Phytophthora ramorum-infected plant material

AIMS: To determine the effects of heat and composting treatments on the viability of the plant pathogen Phytophthora ramorum grown on both artificial and various natural substrates. METHODS AND RESULTS: Phytophthora ramorum was grown on V8 agar, inoculated on bay laurel leaves (Umbellularia californica) and on woody tissues of coast live oak (Quercus agrifolia). Effects on growth, viability and survival were measured as a result of treatment in ovens and compost piles. Direct plating onto PARP medium and pear-baiting techniques were used to determine post-treatment viability. No P. ramorum was recovered at the end of the composting process, regardless of the isolation technique used. By using a PCR assay designed to detect the DNA of P. ramorum, we were able to conclude the pathogen was absent from mature compost and not merely suppressed or dormant. CONCLUSIONS: Some heat and composting treatments eliminate P. ramorum to lower than detectable levels on all substrates tested. SIGNIFICANCE AND IMPACT OF THE STUDY: Composting is an effective treatment option for sanitization of P. ramorum-infected plant material. Assaying for pathogen viability in compost requires a direct test capable of differentiating between pathogen suppression and pathogen elimination.     

Local adaptation to temperature in populations and clonal lineages of the Irish potato famine pathogen Phytophthora infestans

Environmental factors such as temperature strongly impact microbial communities. In the current context of global warming, it is therefore crucial to understand the effects of these factors on human, animal, or plant pathogens. Here, we used a common‐garden experiment to analyze the thermal responses of three life‐history traits (latent period, lesion growth, spore number) in isolates of the potato late blight pathogen Phytophthora infestans from different climatic zones. We also used a fitness index (FI) aggregating these traits into a single parameter. The experiments revealed patterns of local adaptation to temperature for several traits and for the FI, both between populations and within clonal lineages. Local adaptation to temperature could result from selection for increased survival between epidemics, when isolates are exposed to more extreme climatic conditions than during epidemics. We also showed different thermal responses among two clonal lineages sympatric in western Europe, with lower performances of lineage 13_A2 compared to 6_A1, especially at low temperatures. These data therefore stress the importance of thermal adaptation in a widespread, invasive pathogen, where adaptation is usually considered almost exclusively with respect to host plants. This must now be taken into account to explain, and possibly predict, the global distribution of specific lineages and their epidemic potential.     

The hunt for sustainable biocontrol of oomycete plant pathogens,a case study of Phytophthora infestans

Late blight caused by the oomycete Phytophthora infestans is considered to be one of the most severe diseases of potato and tomato worldwide. Whilst current synthetic fungicides are efficient at controlling this disease, they are an environmental and economic burden. In line with EU directives to reduce the use of synthetic pesticides and increase the use of sustainable alternative disease control strategies that can form part of integrated pest management systems, practical biological control solutions are urgently needed. Despite the fact that there has been a large body of scientific research into microorganisms with potential for the biological control of late blight disease, relatively few commercial biocontrol agents, licensed to control late blight, exist. Furthermore, the practical uptake of those in Europe is lower than might be expected, suggesting that such solutions are not yet feasible, or effective. Here we review the scientific literature, focusing on the most recent developments in the hunt for efficient and sustainable biological control of late blight disease. We discuss the progress in our mechanistic understanding of mycoparasite–prey interactions, in the context of late blight and the challenges and limitations to the use of such knowledge in practical disease control within a European context.     

Establishment of a novel virus-induced virulence effector assay for the identification of virulence effectors of plant pathogens using a PVX-based expression vector

Plant pathogens deliver virulence effectors into plant cells to modulate plant immunity and facilitate infection. Although species-specific virulence effector screening approaches have been developed for several pathogens, these assays do not apply to pathogens that cannot be cultured and/or transformed outside of their hosts. Here, we established a rapid and parallel screening assay, called the virus-induced virulence effector (VIVE) assay, to identify putative effectors in various plant pathogens, including unculturable pathogens, using a virus-based expression vector. The VIVE assay uses the potato virus X (PVX) vector to transiently express candidate effector genes of various bacterial and fungal pathogens into Nicotiana benthamiana leaves. Using the VIVE assay, we successfully identified Avh148 as a potential virulence effector of Phytophthora sojae. Plants infected with PVX carrying Avh148 showed strong viral symptoms and high-level Avh148 and viral RNA accumulation. Analysis of P. sojae Avh148 deletion mutants and soybean hairy roots overexpressing Avh148 revealed that Avh148 is required for full pathogen virulence. In addition, the VIVE assay was optimized in N. benthamiana plants at different developmental stages across a range of Agrobacterium cell densities. Overall, we identified six novel virulence effectors from seven pathogens, thus demonstrating the broad effectiveness of the VIVE assay in plant pathology research.     

Susceptibility to Phytophthora ramorum in a key infectious host: landscape variation in host genotype, host phenotype, and environmental factors

Sudden oak death is an emerging forest disease caused by the invasive pathogen Phytophthora ramorum. Genetic and environmental factors affecting susceptibility to P. ramorum in the key inoculum-producing host tree Umbellularia californica (bay laurel) were examined across a heterogeneous landscape in California, USA. Laboratory susceptibility trials were conducted on detached leaves and assessed field disease levels for 97 host trees from 12 225-m(2) plots. Genotype and phenotype characteristics were assessed for each tree. Effects of plot-level environmental conditions (understory microclimate, amount of solar radiation and topographic moisture potential) on disease expression were also evaluated. Susceptibility varied significantly among U. californica trees, with a fivefold difference in leaf lesion size. Lesion size was positively related to leaf area, but not to other phenotypic traits or to field disease level. Genetic diversity was structured at three spatial scales, but primarily among individuals within plots. Lesion size was significantly related to amplified fragment length polymorphism (AFLP) markers, but local environment explained most variation in field disease level. Thus, substantial genetic variation in susceptibility to P. ramorum occurs in its principal foliar host U. californica, but local environment mediates expression of susceptibility in nature.     

Disruption of response regulator gene, devR, leads to attenuation in virulence of Mycobacterium tuberculosis

The devR-devS two-component system of Mycobacterium tuberculosis was identified earlier and partially characterized in our laboratory. A devR::kan mutant of M. tuberculosis was constructed by allelic exchange. The devR mutant strain showed reduced cell-to-cell adherence in comparison to the parental strain in laboratory culture media. This phenotype was reversed on complementation with a wild-type copy of devR. The devR mutant and parental strains grew at equivalent rates within human monocytes either in the absence or in the presence of lymphocytic cells. The expression of DevR was not modulated upon entry of M. tuberculosis into human monocytes. However, guinea pigs infected with the mutant strain showed a significant decrease in gross lesions in lung, liver and spleen; only mild pathological changes in liver and lung; and a nearly 3 log lower bacterial burden in spleen compared to guinea pigs infected with the parental strain. Our results suggest that DevR is required for virulence in guinea pigs but is not essential for entry, survival and multiplication of M. tuberculosis within human monocytes in vitro. The attenuation in virulence of the devR mutant in guinea pigs together with DevR-DevS being a bona fide signal transduction system indicates that DevR plays a critical and regulatory role in the adaptation and survival of M. tuberculosis within tissues.     

Distribution of six virulence factors in Aeromonas species isolated from US drinking water utilities: a PCR identification

AIMS: To examine whether Aeromonas bacteria isolated from municipally treated water had virulence factor genes. METHODS AND RESULTS: A polymerase chain reaction-based genetic characterization determined the presence of six virulence factors genes, elastase (ahyB), lipase (pla/lip/lipH3/alp-1) flagella A and B (flaA and flaB), the enterotoxins, act, alt and ast, in these isolates. New primer sets were designed for all the target genes, except for act. The genes were present in 88% (ahyB), 88% (lip), 59% (fla), 43% (alt), 70% (act) and 30% (ast) of the strains, respectively. Of the 205 isolates tested only one isolate had all the virulence genes. There was a variety of combinations of virulence factors within different strains of the same species. However, a dominant strain having the same set of virulence factors, was usually isolated from any given tap in different rounds of sampling from a single tap. CONCLUSIONS: These results show that Aeromonas bacteria found in drinking water possess a wide variety of virulence-related genes and suggest the importance of examining as many isolates as possible in order to better understand the health risk these bacteria may present. SIGNIFICANCE AND IMPACT OF THE STUDY: This study presents a rapid method for characterizing the virulence factors of Aeromonas bacteria and suggests that municipally treated drinking water is a source of potentially pathogenic Aeromonas bacteria.     

Genome-wide identification of novel genomic islands that contribute to Salmonella virulence in mouse systemic infection

Salmonella pathogenicity islands are inserted into the genome by horizontal gene transfer and are required for expression of full virulence. Here, we performed tRNA scanning of the genome of Salmonella enterica serovar Typhimurium and compared it with that of nonpathogenic Escherichia coli in order to identify genomic islands that contribute to Salmonella virulence. Using deletion analysis, we identified four genomic islands that are required for virulence in the mouse infection model. One of the newly identified pathogenicity islands was the pheV- tRNA-located genomic island, which is comprised of 26 126 bp, and encodes 22 putative genes, including STM3117–STM3138. We also showed that the pheV tRNA-located genomic island is widely distributed among different nontyphoid Salmonella serovars. Furthermore, genes including STM3118–STM3121 were identified as novel virulence-associated genes within the pheV- tRNA-located genomic island. These results indicate that a Salmonella -specific pheV- tRNA genomic island is involved in Salmonella pathogenesis among the nontyphoid Salmonella serovars.     

A specific genetic background is required for acquisition and expression of virulence factors in Escherichia coli

In bacteria, the evolution of pathogenicity seems to be the result of the constant arrival of virulence factors (VFs) into the bacterial genome. However, the integration, retention, and/or expression of these factors may be the result of the interaction between the new arriving genes and the bacterial genomic background. To test this hypothesis, a phylogenetic analysis was done on a collection of 98 Escherichia coli/Shigella strains representing the pathogenic and commensal diversity of the species. The distribution of 17 VFs associated to the different E. coli pathovars was superimposed on the phylogenetic tree. Three major types of VFs can be recognized: (1) VFs that arrive and are expressed in different genetic backgrounds (such as VFs associated with the pathovars of mild chronic diarrhea: enteroaggregative, enteropathogenic, and diffusely-adhering E. coli), (2) VFs that arrive in different genetic backgrounds but are preferentially found, associated with a specific pathology, in only one particular background (such as VFs associated with extraintestinal diseases), and (3) VFs that require a particular genetic background for the arrival and expression of their virulence potential (such as VFs associated with pathovars typical of severe acute diarrhea: enterohemorragic, enterotoxigenic, and enteroinvasive E. coli strains). The possibility of a single arrival of VFs by chance, followed by a vertical transmission, was ruled out by comparing the evolutionary histories of some of these VFs to the strain phylogeny. These evidences suggest that important changes in the genome of E. coli have occurred during the diversification of the species, allowing the virulence factors associated with severe acute diarrhea to arrive in the population. Thus, the E. coli genome seems to be formed by an "ancestral" and a "derived" background, each one responsible for the acquisition and expression of different virulence factors.