Abstract: We have used map-based approaches to clone a locus containing two genes, Avr1b-1 and Avr1b-2, required for avirulence of the oomycete pathogen Phytophthora sojae(Kaufmann & Gerdemann) on soybean plants carrying resistance gene Rps1b. Avr1b-1 was localized to a single 60-kb bacterial artificial chromosome (BAC) clone by finestructure genetic mapping. Avr1b-1 was localized within the 60-kb region by identification of an mRNA that is expressed in a race-specific and infection-specific manner and that encodes a small secreted protein. When the Avr1b-1 protein was synthesized in the yeast Pichia pastoris and the secreted protein infiltrated into soybean leaves, it triggered a hypersensitive response specifically in host plants carrying the Rps1b resistance gene. This response eventually spread to the entire inoculated plant. In some isolates of P. sojae virulent on Rps1b-containing cultivars, such as P7081 (race 25) and P7076 (race 19), the Avr1b-1 gene had numerous substitution mutations indicative of strong divergent selection. In other isolates, such as P6497 (race 2)and P9073 (race 25), there were no substitutions in Avr1b-1, but Avr1b-1 mRNA did not accumulate. Genetic complementation experiments with P6497 revealed the presence of a second gene, Avr1b-2, required for the accumulation of Avr1b-1 mRNA. Avr1b-2 was genetically mapped to the same BAC contig as Avr1b-1, using a cross between P7064 (race 7) and P6497. The Avr1k gene, required for avirulence on soybean cultivars containing Rps1k, was mapped to the same interval as Avr1b-1.
Abstract: Stem and root rot caused by the oomycete pathogen, Phytophthora sojae, is a serious soybean disease. Use of Phytophthora resistance genes (Rps) in soybean cultivars has been very effective in controlling this pathogen. Resistance encoded by Rps genes is manifested through activa-
tion of defense responses. In order to identify candidate signaling genes involved in the expression of Phytophthora resistance in soybean, a cDNA library was prepared from infected etiolated hypocotyl tissues of a Phytophthora resistant soybean cultivar harvested 2 and 4 h following
P. sojae inoculation. In silico subtraction of 101,833 expressed sequence tags (ESTs) originating from unstressed cDNA libraries from 4,737 ESTs of this library resulted in identifcation of 204 genes that were absent in the unstressed libraries. Of the 204 identified genes, seven
were P. sojae genes. Putative function of 91 of the 204 genes could not be assigned based on sequence comparison. Macroarray analyses of all 204 genes led to identification of 60 genes including 15 signaling-related soybean genes and three P. sojae genes, transcripts of which were induced twofold in P. sojae-infected tissues as compared to that in water controls. Eight soybean genes were down-regulated twofold following P. sojae infection as compared to water controls. Differential expression of a few selected genes was confirmed by conducting Northern and RT-PCR analyses. We have shown that two putative regulators of chromosome condensation 1 (RCC1) family proteins were down-regulated in the incompatible interaction. This observation suggested that the nucleocytoplasmic transport function for traYcking protein and non-coding RNA is suppressed during expression of race-specific Phytophthora resistance. Characterization of a cDNA library generated from tissues harvested almost immediately following P. sojae-infection of a resistant cultivar allowed us to identify many candidate signaling genes that are presumably involved in regulating the expression of defense-related pathways for expression of Phytophthora resistance in soybean.
Abstract: The molecular basis of non-host resistance, or species-specific resistance, remains one of the major unknowns in the study of plantmicrobe interactions. In this paper, we describe the characterization of a non-host pathosystem involving the model plant Arabidopsis thaliana and the economically important and destructive oomycete pathogen Phytophthora infestans. Cytological investigations into the early stages of this interaction revealed the germination of P. infestans cysts on Arabidopsis leaves, direct penetration of epidermal cells, formation of infection vesicles and occasionally secondary hyphae, followed by a typical hypersensitive response. P.infestans biomass dynamics during infection of Arabidopsis was monitored using kinetic PCR, revealing an increase in biomass during the first 24 h after inoculation, followed by a decrease in the later stages. Transgenic reporter lines and RNA blot analyses were used to characterize the defence responses induced following P. infestans infection. Significant induction of PDF1.2 was observed at 48 h after inoculation, whereas elevated levels of PR gene expression were detected three days after inoculation. To further characterize this defence response, DNA microarray analyses were carried out to determine the expression profiles for c. 11 000 Arabidopsis cs 16 h after infection. These analyses revealed a significant overlap between Arabidopsis non-host response and other defencerelated treatments described in the literature. In particular, non-host response to P. infestans was clearly associated with activation of the jasmonate pathway. The described Arabidopsis– P.infestans pathosystem offers excellent prospects for improving our understanding of non-host resistance.
Abstract: Late blight, caused by the pathogen Phytophthora infestans, is a devastating disease of potato and tomato, but can also damage other solanaceous hosts. To gain a better understanding of the interaction between P. infestans and these other hosts, the susceptibility of species in three solanaceous genera was investigated. Of the 10 Calibrachoa × hybridus cultivars tested, four were susceptible and six were resistant to the pathogen; susceptible cultivars supported only very limited growth of P. infestans. The majority of the Petunia × hybrida (Petunia) cultivars were susceptible, although less so than susceptible potatoes or tomatoes. Two petunia cultivars displayed differential resistance, suggesting the presence of R genes against P. infestans. The hypersensitive response was present in susceptible, partially resistant, and resistant petunia-P. infestans interactions, but was predominant in the resistant interaction. Young petunias (3 weeks) were more susceptible than older petunias (7 weeks). Nicotiana benthamiana was susceptible to all four P. infestans isolates tested in the lab and became infected during a field epidemic. Several of these isolates were tested for the presence of the inf1 gene, and were found to have and express the gene in vitro. In addition, culture filtrate from these isolates contained 10-kDa proteins and also elicited the hypersensitive response in Nicotiana tabacum and N. benthamiana.
Abstract: Cell death plays a ubiquitous role in plant-microbe interactions, given that it is associated with both susceptible and resistance interactions. A class of cell death–inducing proteins, termed Nep1-like proteins (NLPs), has been reported in bacteria, fungi, and oomycetes. These proteins induce nonspecific necrosis in a variety of dicotyledonous plants. Here, we describe three members of the NLP family from the oomycete Phytophthora infestans (PiNPP1.1, PiNPP1.2, and PiNPP1.3). Using agroinfection with a binary Potato virus X vector, we showed that PiNPP1.1 induces cell death in Nicotiana benthamiana and the host plant tomato. Expression analyses indicated that PiNPP1.1 is up-regulated during late stages of infection of tomato by P. infestans. We compared PiNPP1.1 necrosis-inducing activity to INF1 elicitin, a wellstudied protein that triggers the hypersensitive response in Nicotiana spp. Using virus-induced gene silencing, we showed that the cell death induced by PiNPP1.1 is dependent on the ubiquitin ligase–associated protein SGT1 and the heat-shock protein HSP90. In addition, cell death triggered by PiNPP1.1 but not that by INF1 was dependent on the defense-signaling proteins COI1, MEK2, NPR1, and TGA2.2, suggesting distinct signaling requirements. Combined expression of PiNPP1.1 and INF1 in N. benthamiana resulted in enhanced cell death, suggesting synergistic interplay between the two cell-death responses. Altogether, these results point to potentially distinct but interacting cell-death pathways induced by PiNPP1.1 and INF1 in plants.
Abstract:Leptosphaeria maculans,the causal agent of stem canker of oilseed rape (Brassica napus), develops gene-for-gene interactions with oilseed rape, and four L. maculans avirulence (AVR) genes (AvrLm1, AvrLm2,
AvrLm4, and alm1) were previously genetically characterized. Based on the analysis of progeny of numerous in vitro crosses between L. maculans isolates showing either already characterized or new differential
interactions, this work aims to provide an overview of the AVR genes
that may specify incompatibility toward B. napus and the related species
B. juncea and B. rapa. Two novel differential interactions were thus identified between L. maculans and B. napus genotypes, one of themcorresponding to a complete resistance to European races of L. maculans. In both cases, a single gene control of avirulence was established (genes AvrLm3 and AvrLm7). Similarly, a single gene control of avirulence toward a B. rapa genotype, also resistant to European L. maculans isolates, was demonstrated (gene AvrLm8). Finally, a digenic control of avirulence toward B. juncea was established (genes AvrLm5 and AvrLm6). Linkage analyses demonstrated that at least four unlinked L. maculansgenomic regions, including at least one AVR gene cluster (AvrLm1-AvrLm2-AvrLm6), are involved in host specificity. The AvrLm3-AvrLm4-AvrLm7 region may correspond either to a second AVR gene cluster or to a multiallelic AVR gene.
Abstract:Phytophthora spp. secrete proteins called elicitins in vitro that can specifically induce hypersensitive response and systemic acquired resistance in tobacco. In Phytophthora nicotianae parasitica, the causal agent of black shank, most isolates virulent on tobacco are unable to produce elicitins in vitro. Recently, however, a few elicitin-producing P. parasitica strains virulent on tobacco have been isolated. We investigated the potential diversity of elicitin genes in P. parasitica isolates belonging to different genotypes and with various virulence levels toward tobacco as well as elicitin expression pattern in vitro and in planta. Although elicitins are encoded by a multigene family, parA1 is the main elicitin gene expressed. This gene is highly conserved among isolates, regardless of the elicitin production and virulence levels toward tobacco. Moreover, we show that elicitin-producing P. parasitica isolates virulent on tobacco down regulate parA1 expression during compatible interactions, whichever host plant is tested. Conversely, one elicitinproducing P. parasitica isolate that is pathogenic on tomato and avirulent on tobacco still expresses parA1 in the compatible interaction. Therefore, some P. parasitica isolates may evade tobacco recognition by down regulating parA1 in planta. The in planta down regulation of parA1 may constitute a suitable mechanism for P. parasitica to infect tobacco without deleterious consequences for the pathogen.
Abstract: 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 ofwhich are in themature 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 conservedmotif 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.
Abstract: Suppression subtractive hybridization was used to isolate the genes which are specifically up-regulated in the biotrophic phase of the incompatible interaction between a potato genotype, 1512c(16), containing the resistance gene R2, and a Phytophthora infestansisolate containing the avirulence gene Avr2. Eight cDNAs were upregulated in the biotrophic phase of the incompatible interaction. Seven of these were also up-regulated in the compatible interaction,but not until late in the necrotrophic phase. Amongst the sequences to be isolated were genes encoding the cysteine protease cathepsin
B, StCathB, and an oxysterol binding protein, StOBP1; equivalent
genes are involved in programmed cell death (PCD) processes in animals, but have yet to be implicated in such processes in plants. Whereas StOBP1
was up-regulated early in potato plants containing either R gene-mediated or moderate to high levels of field resistance, the highest levels of up-regulation of StCathB were observed early in R gene-mediated resistance but gradually increased from the early to late stages of field resistance, revealing these genes to be components of independent defence pathways and providing a means of distinguishing between these forms of resistance. StOBP1 was up-regulated by oligogalacturonides (plant cell wall breakdown products generated by pectinase activities),indicating that it is also a component of a general, non-specific defence pathway and is unlikely to play a role in PCD. In contrast, the expression of StCathB was unaffected by oligogalacturonide treatment, further associating its up-regulation specifically with the gene-for-gene interaction.
Abstract: Bacterial, oomycete and fungal plant pathogens establish disease by translocation of effector proteins into host cells, where they may directly manipulate host innate immunity. In bacteria, translocation is through the type III secretion system, but analogous processes for effector delivery are uncharacterized in fungi and oomycetes. Here we report functional analyses of two motifs,RXLR and EER, present in translocated oomycete effectors.We use the Phytophthora infestansRXLR-EER-containing protein Avr3a as a reporter for translocation because it triggers RXLR-EER-independent hypersensitive cell death following recognition within plant cells that contain the R3a resistance protein4,5. We show that Avr3a,with orwithout RXLR-EER motifs, is secreted from P. infestans biotrophic structures called haustoria, demonstrating that these motifs are not required for targeting to haustoria or for secretion. However, following replacement of Avr3aRXLR-EER motifswith alanine residues, singly or in combination, or with residues KMIK-DDK—representing a change that conserves physicochemical properties of the protein—P. infestans fails to deliver Avr3a or an Avr3a–GUS fusion protein into plant cells, demonstrating that these motifs are required for translocation. We show that RXLR-EER-encoding genes are transcriptionally upregulated during infection. Bioinformatic analysis identifies 425 potential genes encoding secreted RXLR-EER class proteins in the P. infestans genome. Identification of this class of proteins provides unparalleled opportunities to determine how oomycetes manipulate hosts to establish infection.
Abstract