Abstract: he downy mildew (Hyaloperonospora parasitica) effector proteins ATR1 and ATR13 trigger RPP1-Nd/WsB– and RPP13-Nd–
dependent resistance, respectively, in Arabidopsis thaliana. To better understand the functions of these effectors during compatible and incompatible interactions of H. parasitica isolates on Arabidopsis accessions, we developed a novel delivery system using Pseudomonas syringae type III secretion via fusions of ATRs to the N terminus of the P. syringae
effector protein, AvrRPS4. ATR1 and ATR13 both triggered the hypersensitive response (HR) and resistance to bacterial
pathogens in Arabidopsis carrying RPP1-Nd/WsB or RPP13-Nd, respectively, when delivered from P. syringae pv tomato (Pst) DC3000. In addition, multiple alleles of ATR1 and ATR13 confer enhanced virulence to Pst DC3000 on susceptible Arabidopsis accessions. We conclude that ATR1 and ATR13 positively contribute to pathogen virulence inside host cells. Two ATR13 alleles suppressed bacterial PAMP (for Pathogen-Associated Molecular Patterns)-triggered callose deposition in susceptible Arabidopsis when delivered by DC3000 DCEL mutants. Furthermore, expression of another allele of ATR13 in plant cells suppressed PAMP-triggered reactive oxygen species production in addition to callose deposition. Intriguingly, although Wassilewskija (Ws-0) is highly susceptible to H. parasitica isolate Emco5, ATR13Emco5 when delivered by Pst DC3000 triggered localized immunity, including HR, on Ws-0. We suggest that an additional H. parasitica Emco5 effector might suppress ATR13-triggered immunity.
Abstract: A diverse range of plant proteases are implicated in pathogen perception and in subsequent signalling and execution of disease resistance. We demonstrate, using protease inhibitors and virus-induced gene silencing(VIGS), that the plant papain cysteine protease cathepsin B is required for the disease resistance hypersensitive response (HR). VIGS of cathepsin B prevented programmed cell death (PCD) and compromised disease resistance induced by two distinct non-host bacterial pathogens. It also suppressed the HR triggered by transient co-expression of potato R3a and Phytophthora infestans Avr3a genes. However, VIGS of cathepsin B did not compromise HR following recognition of Cladosporium fulvum AVR4 by tomato Cf-4, indicating that plant PCD can be independent of cathepsin B. The non-host HR to Erwinia amylovora was accompanied by a transient increase in cathepsin B transcript level and enzymatic activity and induction of the HR marker gene Hsr203. VIGS of cathepsin B significantly reduced the induction of Hsr203 following E. amylovora challenge, further demonstrating a role for this protease in PCD. Whereas cathepsin B is often relocalized from the lysosome to the cytosol during animal PCD, plant cathepsin B is secreted into the apoplast, and is activated upon secretion in the absence of pathogen challenge.
Abstract: A total of 23 Scottish and 14 Dutch potato R-gene differentials as well as five Austrian, two Dutch and two German commercial potato cultivars were screened for the R1 allele conferring resistance to Phytophthora infestans carrying Avr1, via PCR amplification and sequencing. A single 1400 bp fragment with complete sequence identity to the corresponding part of the R1 allele, was obtained from genomic DNA of all potato R-gene differential clones whose denomination indicates R1 or a combination of R1 and other major resistance factors. The R1 allele was detected, as expected,in all these clones. This fragment also occurred in one Austrian and one German cultivar. Unexpectedly, the same R1 allele also was detected within all R5, R6 and R9 differentials.
Abstract: To gain deeper understanding of the host-pathogen interaction in the system potato- Phytophthora infestans, subtractive hybridization in combination with cDNA array hybridization was used. Leaflets of a moderately resistant and a susceptible potato cultivar were inoculated with P. infestans. The infection of the potato leaves was quantified by real-time quantitative PCR. Using infected and control tissue, two cDNA libraries highly enriched for P. infestans-induced genes were prepared. Within 531 clones randomly picked and sequenced from the libraries, 285 unigenes were found, from which 182 clones were selected for further analysis by cDNA array hybridization. Sixteen hours post inoculation genes were not induced significantly, whereas 72 h post inoculation induction of gene expression was clearly detectable. In both cultivars, 143 genes were induced moderately (≥ two-fold), and 35 of the selected genes appeared to be strongly induced (≥ seven-fold). Among these clones were mainly genes associated with stress and/or defence mechanisms. The strongest gene induction was found in 4-week-old susceptible plants. In the moderately resistant cultivar,transcripts of a number of genes accumulate with plant age; as a result, induction of gene expression upon infection was less pronounced. Down-regulation of three genes was observed in both cultivars, upon infection. Transcript levels of these three genes increased in uninfected plants within 4 weeks of growth. Other differences in defence responses of the two cultivars could be determined and their effects are discussed.
Abstract: Recognition is the earliest step in any direct plant-microbe interaction.
Recognition between Phytophthora pathogens, which are oomycetes, phylogenetically distinct from fungi, has been studied at two levels. Recognition of the host by the pathogen has focused on recognition of chemical, electrical, and physical features of plant roots by zoospores. Both host-specific factors such as isoflavones, and host-nonspecific factors such as amino acids, calcium, and electrical fields, influence zoospore taxis, encystment, cyst germination, and hyphal chemotropism in guiding the pathogen to potential infection sites. Recognition of the pathogen by the host defense machinery has been analyzed using biochemical and genetic approaches. Biochemical approaches have identified chemical elicitors of host defense responses, and in some cases, their cognate receptors from the host. Some elicitors, such as glucans and fatty acids, have broad host ranges, whereas others such as elicitins have narrow host ranges. Most elicitors identified appear to contribute primarily to basic or nonhost resistance. Genetic analysis has identified host resistance (R) genes and pathogen avirulence (Avr)genes that interact in a gene-for-genemanner. One Phytophthora Avr gene, Avr1b from P. sojae, has been cloned and characterized. It encodes a secreted elicitor that triggers a system-wide defense response in soybean plants carrying the cognate R gene, Rps1b.
Abstract: Suppression Subtractive Hybridization (SSH) was used to search for genes of Phytophthora infestans that are induced during the infection of potato. To avoid having to distinguish the genes of the pathogen from the plant genes involved in defence responses and to isolate the genes involved in the early stages of interaction, mycelium of P. infestans was induced by contact with the host plant and then separated from the plant tissue. A differential cDNA library was generated by SSH that compared such induced mycelium with mycelium incubated in water. The expression of about 100 cDNA fragments from this differential cDNA library was analysed by hybridization of the arrayed PCR products with mRNA from control and induced mycelium. Twenty per cent of them showed increased transcript levels in mycelium within the first 24 h after exposure to a potato leaf. For six of these cDNA clones the elevated expression in response to the potato leaf could be proven by RNA gel blot analysis. Five of these cDNA clones have predicted amino acid sequence homologies to entries in the databases, including an amino acid transporter, a sucrose transporter, a spliceosome-associated factor, an ABC transporter, and a cell division control protein. We showed that the genes corresponding to these six cDNA clones are differentially regulated during their life. Reliable gene expression analysis of Phytophthora in infected leaf tissue is not possible until c. 48 h post-infection, but for two of the genes we identified, induction during in planta growth was detectable by RNA gel blot analysis. Therefore the SSH library that we have created provides a basis for the identification of P. infestans genes that are up-regulated during the interaction with the plant, which could be important for pathogenicity.
Abstract: Suppression Subtractive Hybridization (SSH) was applied in a search for genes induced during the compatible interaction between Phytophthora infestans and potato. Using potato leaves that had been treated with benzo (1,2,3)thiadiazole-7-carbothioic acid S-methylester (BTH) as the control tissue, a low redundancy library with a relatively low frequency of the classic
plant Pathogenesis-Related (PR) genes was generated. 288 of the clones were screened for induced sequences using Inverse Northern analysis (hybridizing the arrayed clones with radiolabelled cDNA populations). Of the 75 clones that were detectable by this method, 43 appeared to be induced. Eleven of these clones were then analysed by total RNA blot analysis, and
elevation of transcript levels during P. infestans infection was
confirmed for 10 of them. Some of the cDNAs analysed by RNA blot analysis have homology to genes already known to be induced during infection, e.g. to β-1,3-glucanase. Another group of cDNAs have homology to enzymes involved in detoxification: gamma-glutamylcysteine synthetase, cytochrome P450, glutathione S-transferase and an MRP-type ABC transporter. Other infection induced cDNAs encode putative proteins that have not previously been reported to be induced by infection: e.g. the ER-located chaperone BiP, and a homologue of Aspergillusnidulans SudD, which was isolated as a suppressor of a mutation in chromosome disjunction. The differential library therefore presents the opportunity to analyse the metabolic changes occurring during infection, and the disease process itself in more detail.
Abstract: Oomycete plant pathogens deliver effector proteins inside host cells tomodulate plant defense circuitry and toenable parasitic colonization. These effectors are defined by a conservedmotif, termed RXLR (for Arg, any amino acid, Leu, Arg), that is located downstream of the signal peptide and that has been implicated in host translocation. Because the phenotypes of RXLR
effectors extend to plant cells, their genes are expected to be the direct target of the evolutionary forces that drive the antagonistic interplay between pathogen and host. We used the draft genome sequences of three oomycete plant pathogens,Phytophthora sojae, Phytophthora ramorum, and Hyaloperonospora parasitica, to generate genome-wide catalogs of RXLR effector genes and determine the extent to which these genes are under positive selection. These analyses revealed that the RXLR sequence is overrepresented and positionally constrained in the secretome of Phytophthora relative to other eukaryotes. The three examined plant pathogenic oomycetes carry complex and diverse sets of RXLR effector genes that have undergone relatively rapid birth and death evolution.We obtained robust evidence of positive selection inmore than two-thirds of the examined paralog families of RXLR effectors. Positive selection has acted for the most part on the C-terminal region, consistentwith the view that RXLR effectors aremodular, with the N terminus involved in secretion and host translocation and the C-terminal domain dedicated to modulating host defenses inside plant cells.
Abstract: Fungi and oomycetes are the causal agents of many of the worlds most serious plant diseases and are unique among the microbial pathogens in being able to breach the intact surfaces of host plants, rapidly establishing infections that can have disastrous consequences for large-scale agricultural production. The ability to cause plant disease is not a common trait among the many saprotrophic and mutualistic fungal species, but it is a very widespread one, occurring throughout the fungal kingdom (James et al., 2006). Recently, there have been a number of studies published describing the genome sequences of a diverse set of fungi and oomycetes (Table 1), including one published in this issue of The Plant Cell (Hane et al., 2007), and this provides an opportunity to review what we have learned so far from sequencing the genomes of pathogenic and free-living fungi and also to look forward to the mass of genome sequence information that is likely to be generated in the next few years. The deployment of low-cost, high-throughput DNA sequencing technologies and large-scale functional genomics to eukaryotic plant pathogens will provide new insight into their biology and into the evolution of pathogenicity.
Abstract: The cellulose binding elicitor lectin (CBEL) from Phytophthora parasitica nicotianae contains two cellulose binding domains (CBDs) belonging to the Carbohydrate Binding Module1 family, which is found almost exclusively in fungi. Themechanism by which CBEL is perceived by the host plant remains unknown. The role of CBDs in eliciting activity was investigated using modified versions of the protein produced in Escherichia coli or synthesized in planta through the potato virus X expression
system. Recombinant CBEL produced by E. coli elicited necrotic lesions and defense gene expression when injected into tobacco (Nicotiana tabacum) leaves. CBEL production in planta induced necrosis. Site-directed mutagenesis on aromatic amino acid residues located within the CBDs as well as leaf infiltration assays using mutated and truncated recombinant proteins confirmed the importance of intact CBDs to induce defense responses. Tobacco and Arabidopsis thaliana leaf infiltration assays using synthetic peptides showed that the CBDs of CBEL are essential and sufficient to stimulate defense responses. Moreover, CBEL elicits a transient variation of cytosolic calcium levels in tobacco cells but not in protoplasts. These results defene CBDs as a novel class ofmolecular patterns in oomycetes that are targeted by the innate immune system
of plants and might act through interaction with the cell wall.
Abstract