Late Blight Pathogen Phytophthora Infestans Research Articles

Differential gene induction in resistant and susceptible potato cultivars at early stages of infection by Phytophthora infestans

Sarpo Mira, a potato variety with high resistance against the late blight pathogen Phytophthora infestans, is being used in breeding programs to increase late blight resistance in commercial varieties. Discovering genes that are important for P. infestans resistance will assist in the development of molecular markers for the selection of new resistant cultivars and the use of resistant varieties will reduce the environmental, health and financial costs associated with the use of pesticides. Using complementary DNA amplified fragment length polymorphism analyses, differentially expressed genes involved in the potato-P. infestans interaction were identified in the susceptible Bintje and in the resistant Sarpo Mira potato cultivars. Forty-eight differentially expressed transcript derived fragments (TDFs) were cloned and sequenced. The expression profiles of some of these genes were analyzed in detail using quantitative RT-PCR at seven time points: 1, 4, 17, 24, 30, 41 and 65 hours after inoculation (hai). We found that five transcripts with homologies to pathogenesis/defense-related genes and two TDFs with homology to transcription factors were significantly induced to higher levels in the resistant cultivar at very early stages of the infection (1 hai). Interestingly, most of these genes showed different expression profiles throughout the whole infection process between both cultivars. Particularly during its biotrophic growth phase, P. infestans triggered the down-regulation of infection responsive genes in the susceptible but not in the resistance cultivar. Our results suggest that these newly identified early-induced transcripts may be good candidates for conferring Sarpo Mira's resistance to late blight and they could be useful molecular markers for the selection of new resistant cultivars.

Presence/absence, differential expression and sequence polymorphisms between PiAVR2 and PiAVR2‐like in Phytophthora infestans determine virulence on R2 plants

• A detailed molecular understanding of how oomycete plant pathogens evade disease resistance is essential to inform the deployment of durable resistance (R) genes. • Map-based cloning, transient expression in planta, pathogen transformation and DNA sequence variation across diverse isolates were used to identify and characterize PiAVR2 from potato late blight pathogen Phytophthora infestans. • PiAVR2 is an RXLR-EER effector that is up-regulated during infection, accumulates at the site of haustoria formation, and is recognized inside host cells by potato protein R2. Expression of PiAVR2 in a virulent P. infestans isolate conveys a gain-of-avirulence phenotype, indicating that this is a dominant gene triggering R2-dependent disease resistance. PiAVR2 presence/absence polymorphisms and differential transcription explain virulence on R2 plants. Isolates infecting R2 plants express PiAVR2-like, which evades recognition by R2. PiAVR2 and PiAVR2-like differ in 13 amino acids, eight of which are in the C-terminal effector domain; one or more of these determines recognition by R2. Nevertheless, few polymorphisms were observed within each gene in pathogen isolates, suggesting limited selection pressure for change within PiAVR2 and PiAVR2-like. • Our results direct a search for R genes recognizing PiAVR2-like, which, deployed with R2, may exert strong selection pressure against the P. infestans population.

The receptor-like kinase SERK3/BAK1 is required for basal resistance against the late blight pathogen phytophthora infestans in Nicotiana benthamiana.

BackgroundThe filamentous oomycete plant pathogen Phytophthora infestans causes late blight, an economically important disease, on members of the nightshade family (Solanaceae), such as the crop plants potato and tomato. The related plant Nicotiana benthamiana is a model system to study plant-pathogen interactions, and the susceptibility of N. benthamiana to Phytophthora species varies from susceptible to resistant. Little is known about the extent to which plant basal immunity, mediated by membrane receptors that recognise conserved pathogen-associated molecular patterns (PAMPs), contributes to P. infestans resistance.Principal FindingsWe found that different species of Phytophthora have varying degrees of virulence on N. benthamiana ranging from avirulence (incompatible interaction) to moderate virulence through to full aggressiveness. The leucine-rich repeat receptor-like kinase (LRR-RLK) BAK1/SERK3 is a major modulator of PAMP-triggered immunity (PTI) in Arabidopsis thaliana and N. benthamiana. We cloned two NbSerk3 homologs, NbSerk3A and NbSerk3B, from N. benthamiana based on sequence similarity to the A. thaliana gene. N. benthamiana plants silenced for NbSerk3 showed markedly enhanced susceptibility to P. infestans infection but were not altered in resistance to Phytophthora mirabilis, a sister species of P. infestans that specializes on a different host plant. Furthermore, silencing of NbSerk3 reduced the cell death response triggered by the INF1, a secreted P. infestans protein with features of PAMPs.Conclusions/SignificanceWe demonstrated that N. benthamiana NbSERK3 significantly contributes to resistance to P. infestans and regulates the immune responses triggered by the P. infestans PAMP protein INF1. In the future, the identification of novel surface receptors that associate with NbSERK3A and/or NbSERK3B should lead to the identification of new receptors that mediate recognition of oomycete PAMPs, such as INF1.

At the Frontier; RXLR Effectors Crossing the Phytophthora?Host Interface

Plants are constantly beset by pathogenic organisms. To successfully infect their hosts, plant pathogens secrete effector proteins, many of which are translocated to the inside of the host cell where they manipulate normal physiological processes and undermine host defense. The way by which effectors cross the frontier to reach the inside of the host cell varies among different classes of pathogens. For oomycete plant pathogens – like the potato late blight pathogen Phytophthora infestans – it has been shown that effector translocation to the host cell cytoplasm is dependent on conserved amino acid motifs that are present in the N-terminal part of effector proteins. One of these motifs, known as the RXLR motif, has a strong resemblance with a host translocation motif found in effectors secreted by Plasmodium species. These malaria parasites, that reside inside specialized vacuoles in red blood cells, make use of a specific protein translocation complex to export effectors from the vacuole into the red blood cell. Whether or not also oomycete RXLR effectors require a translocation complex to cross the frontier is still under investigation. For one P. infestans RXLR effector named IPI-O we have found a potential host target that could play a role in establishing the first contact between this effector and the host cell. This membrane spanning lectin receptor kinase, LecRK-I.9, interacts with IPI-O via the tripeptide RGD that overlaps with the RXLR motif. In animals, RGD is a well-known cell adhesion motif; it binds to integrins, which are membrane receptors that regulate many cellular processes and which can be hijacked by pathogens for either effector translocation or pathogen entry into host cells.

An Effector-Targeted Protease Contributes to Defense against Phytophthora infestans and Is under Diversifying Selection in Natural Hosts

Since the leaf apoplast is a primary habitat for many plant pathogens, apoplastic proteins are potent, ancient targets for apoplastic effectors secreted by plant pathogens. So far, however, only a few apoplastic effector targets have been identified and characterized. Here, we discovered that the papain-like cysteine protease C14 is a new common target of EPIC1 and EPIC2B, two apoplastic, cystatin-like proteins secreted by the potato (Solanum tuberosum) late blight pathogen Phytophthora infestans. C14 is a secreted protease of tomato (Solanum lycopersicum) and potato typified by a carboxyl-terminal granulin domain. The EPIC-C14 interaction occurs at a wide pH range and is stronger than the previously described interactions of EPICs with tomato defense proteases PIP1 and RCR3. The selectivity of the EPICs is also different when compared with the AVR2 effector of the fungal tomato pathogen Cladosporium fulvum, which targets PIP1 and RCR3, and only at apoplastic pH. Importantly, silencing of C14 increased susceptibility to P. infestans, demonstrating that this protease plays a role in pathogen defense. Although C14 is under conservative selection in tomato, it is under diversifying selection in wild potato species (Solanum demissum, Solanum verrucosum, and Solanum stoliniferum) that are the natural hosts of P. infestans. These data reveal a novel effector target in the apoplast that contributes to immunity and is under diversifying selection, but only in the natural host of the pathogen.

Enhanced defense responses of tomato plants against late blight pathogen Phytophthora infestans by pre-inoculation with rhizobacteria

The efficacy of resistance in tomato plants, induced by rhizobacteria isolated from Jeju Island in Korea, against late blight disease caused by Phytophthora infestans was tested. Among the bacterial isolates tested, pre-inoculation with four isolates induced an effective defense against late blight. The four isolates, TRH423-3, TRH427-2, KRJ502-1 and KRY505-3, were identified as Burkholderia gladioli, Miamiensis avidus, Acinetobacter quenomosp and Bacillus cereus, respectively, by sequencing the ribosomal intergenic spacer region. They also promoted the growth of tomato seedlings. To illustrate resistance mechanisms, the infection process by P. infestans was examined using a fluorescence microscope. There were no noticeable differences in the rate of germination and appressorium formation between the untreated and pre-inoculated plants. However, callose was more frequently formed at the penetration sites on the leaves of pre-inoculated plants than the untreated plants, suggesting that these rhizobacterial isolates induce defense responses against P. infestans.

Novel potato C2H2-type zinc finger protein gene, StZFP1, which responds to biotic and abiotic stress, plays a role in salt tolerance

Many TFIIIA-type zinc finger proteins (ZFPs) play important roles in stress responses in plants. In the present study, a novel zinc finger protein gene, StZFP1, was cloned from potato. StZFP1 is a typical TFIIIA-type two-finger zinc finger gene with one B-box domain, one L-box domain and a DLN-box/EAR motif. The StZFP1 genes belong to a small gene family with an estimated copy number of four or five, located on chromosome I. StZFP1 is constitutively expressed in leaves, stems, roots, tubers and flowers of adult plants. Expression of StZFP1 can be induced by salt, dehydration and exogenously applied ABA. StZFP1 expression is also responsive to infection by the late blight pathogen Phytophthora infestans. Transient expression analysis of StZFP1:GFP fusion protein revealed that StZFP1 is preferentially localised in the nucleus. Ectopic expression of StZFP1, driven by the Arabidopsis rd29A promoter in transgenic tobacco, increased plant tolerance to salt stress. These results demonstrate that StZFP1 might be involved in potato responses to salt and dehydration stresses through an ABA-dependent pathway.

Effect of Different Genotypes of Phytophthora infestans (Mont. de Bary) and Temperature on Tuber Disease Development

The interactions of different cultivars/Advance Breeding Lines (ABL) of potato with different genotypes of the potato late blight pathogen (Phytophthora infestans) at three storage temperatures on tuber late blight development were evaluated. The contribution of the medullar storage tissues was assessed rather than the periderm and outer cortical cell tissue. Tuber late blight severity measured as tuber darkening [mean Relative Average Reflectance Intensity [RARI (%)] generally increased with temperature. There was little difference in tuber late blight development between 7°C and 10°C treatments and in some combinations significantly more tissue darkening developed at 7 than at 10°C but little or no development occurred at 3°C. Resistance in tubers was observed only in Torridon and Stirling and to some extent Jacqueline Lee, but the cultivar Missaukee had weak tuber resistance. The US-8 genotype isolates were the most aggressive in tubers in most years causing rapid and significantly more tuber damage than any other genotype of P. infestans and similar to the US-6, US-10 and US-14 isolates used in 2006.

Dl-β-Aminobutyric Acid–Induced Resistance of Potato Against Phytophthora infestans Requires Salicylic Acid but Not Oxylipins

Inducing systemic resistance responses in crop plants is a promising alternative way of disease management. To understand the underlying signaling events leading to induced resistance, functional analyses of plants defective in defined signaling pathway steps are required. We used potato, one of the economically most-important crop plants worldwide, to examine systemic resistance against the devastating late blight pathogen Phytophthora infestans, induced by treatment with dl-beta-aminobutyric acid (BABA). Transgenic plants impaired in either the 9-lipoxygenase pathway, which produces defense-related compounds, or the 13-lipoxygenase pathway, which generates jasmonic acid-derived signals, expressed wild-type levels of BABA-induced resistance. Plants incapable of accumulating salicylic acid (SA), on the other hand, failed to mount this type of induced resistance. Consistently, treatment of these plants with the SA analog 2,6-dichloroisonicotinic acid restored BABA-induced resistance. Together, these results demonstrate the indispensability of a functional SA pathway for systemic resistance in potato induced by BABA.

Fitness costs associated with unnecessary virulence factors and life history traits: evolutionary insights from the potato late blight pathogen Phytophthora infestans

BackgroundIn gene-for-gene models of plant-pathogen interactions, the existence of fitness costs associated with unnecessary virulence factors still represents an issue, both in evolutionary biology and agricultural sciences. Measuring such costs experimentally has proven difficult, especially in pathogens not readily amenable to genetic transformation, since the creation of isogenic lines differing only by the presence or absence of avirulence genes cannot be achieved in many organisms. Here, we circumvented this difficulty by comparing fitness traits in groups of Phytophthora infestans isolates sharing the same multilocus fingerprint, but differing by their virulence/avirulence spectrum.ResultsFitness was assessed from calculations derived from the basic reproduction number, combining several life history traits (latent period, spore density and lesion growth rate) evaluated on leaflets of the potato cultivar Bintje, which is free of resistance genes. A statistically significant fitness cost was found in isolates virulent to the R10 resistance gene. That cost was due to a lower spore production in virulent isolates; however, the latent period was shorter in virulent isolates. Similar trends, although not statistically significant, were observed for the other genes tested.ConclusionThe data likely reflect the adaptive response of the pathogen to the cost associated with virulence. They suggest strong trade-offs between life history traits related to pathogenicity and adaptive biology of pathogens.

Molecular mechanisms of generation for nitric oxide and reactive oxygen species, and role of the radical burst in plant immunity.

Rapid production of nitric oxide (NO) and reactive oxygen species (ROS) has been implicated in the regulation of innate immunity in plants. A potato calcium-dependent protein kinase (StCDPK5) activates an NADPH oxidase StRBOHA to D by direct phosphorylation of N-terminal regions, and heterologous expression of StCDPK5 and StRBOHs in Nicotiana benthamiana results in oxidative burst. The transgenic potato plants that carry a constitutively active StCDPK5 driven by a pathogen-inducible promoter of the potato showed high resistance to late blight pathogen Phytophthora infestans accompanied by HR-like cell death and H(2)O(2) accumulation in the attacked cells. In contrast, these plants showed high susceptibility to early blight necrotrophic pathogen Alternaria solani, suggesting that oxidative burst confers high resistance to biotrophic pathogen, but high susceptibility to necrotrophic pathogen. NO and ROS synergistically function in defense responses. Two MAPK cascades, MEK2-SIPK and cytokinesis-related MEK1-NTF6, are involved in the induction of NbRBOHB gene in N. benthamiana. On the other hand, NO burst is regulated by the MEK2-SIPK cascade. Conditional activation of SIPK in potato plants induces oxidative and NO bursts, and confers resistance to both biotrophic and necrotrophic pathogens, indicating the plants may have obtained during evolution the signaling pathway which regulates both NO and ROS production to adapt to wide-spectrum pathogens.

UNRAVELING THE SENSES OF PHYTOPHTHORA; LEADS TO NOVEL CONTROL STRATEGIES?

Oomycetes cause devastating diseases on plants and animals. They cause major yield losses in many crop plants and their control heavily depends on agrochemicals. This is certainly true for the potato late blight pathogen Phytophthora infestans. Strong concerns about adverse effects of agrochemicals on food safety and environment are incentives for the development of novel, environmental friendly control strategies preferably based on natural products. Cyclic lipopeptides (CLPs) were recently discovered as a new class of natural compounds with strong activities against oomycetes including Phytophthora. CLPs lyse zoospores, inhibit mycelial growth and effectively reduce late blight disease. In order to unravel how Phytophthora senses CLPs and other environmental signals we follow two approaches. On the one hand, we monitor genome wide changes in gene expression induced by CLPs with the aim to identify the cellular pathways targeted by CLPs. On the other hand, we analyse components of ubiquitous signal transduction pathways with the aim to identify features that are unique for Phytophthora or oomycetes and, hence, could be suitable targets for novel anti-oomycete agents. Mining and comparing whole genome sequences have revealed that Phytophthora harbours many novel phospholipid modifying enzymes, unique for oomycetes. They have aberrant combinations of catalytic and regulatory domains occasionally combined with transmembrane domains. The latter resemble receptors that might be activated by extracellular ligands. Phospholipids, the substrates of these enzymes, are structural membrane components that also function in signalling. Together these findings open new avenues of research aimed at target-discovery in oomycetes.

Correlation Between Transcript Abundance of the RB Gene and the Level of the RB-Mediated Late Blight Resistance in Potato

Numerous disease-resistance genes have been cloned and characterized in various plant species. Only a few of these reported genes were transcriptionally induced or had enhanced transcription upon pathogen infection. Here, we report that transcription of the RB gene, which was cloned from the wild potato species Solanum bulbocastanum and confers resistance to potato late blight, was significantly increased after inoculation with the late blight pathogen Phytophthora infestans. Different RB transgenic lines showed different levels of resistance, which were correlated with the amounts of RB transcript in the transgenic plants. Different transgenic lines also showed different patterns of RB transcription 1, 3, and 5 days after P. infestans inoculation. Interestingly, the RB gene showed a higher basal level of transcription and a more dramatic transcriptional increase upon inoculation in S. bulbocastanum than in all potato transgenic lines. Our results revealed a predictive correlation between transcript abundance of the RB gene and the level of the RB-mediated late blight resistance. High level of resistance was associated with a combination of rapid RB transcript induction immediately after pathogen infection followed by the steady production of RB transcript. Thus, the transcription level of the RB gene provides a valuable marker for selecting and deploying RB-containing potato lines for late blight control.

The chloroplast protein RPH1 plays a role in the immune response of Arabidopsis to Phytophthora brassicae

Plant immune responses to pathogens are often associated with enhanced production of reactive oxygen species (ROS), known as the oxidative burst, and with rapid hypersensitive host cell death (the hypersensitive response, HR) at sites of attempted infection. It is generally accepted that the oxidative burst acts as a promotive signal for HR, and that HR is highly correlated with efficient disease resistance. We have identified the Arabidopsis mutant rph1 (resistance to Phytophthora 1), which is susceptible to the oomycete pathogen Phytophthora brassicae despite rapid induction of HR. The susceptibility of rph1 was specific for P. brassicae and coincided with a reduced oxidative burst, a runaway cell-death response, and failure to properly activate the expression of defence-related genes. From these results, we conclude that, in the immune response to P. brassicae, (i) HR is not sufficient to stop the pathogen, (ii) HR initiation can occur in the absence of a major oxidative burst, (iii) the oxidative burst plays a role in limiting the spread of cell death, and (iv) RPH1 is a positive regulator of the P. brassicae-induced oxidative burst and enhanced expression of defence-related genes. Surprisingly, RPH1 encodes an evolutionary highly conserved chloroplast protein, indicating a function of this organelle in activation of a subset of immune reactions in response to P. brassicae. The disease resistance-related role of RPH1 was not limited to the Arabidopsis model system. Silencing of the potato homolog StRPH1 in a resistant potato cultivar caused susceptibility to the late blight pathogen Phytophthora infestans.

Towards understanding the virulence functions of RXLR effectors of the oomycete plant pathogen Phytophthora infestans

Plant pathogens establish infection by secretion of effector proteins that may be delivered inside host cells to manipulate innate immunity. It is increasingly apparent that the ubiquitin proteasome system (UPS) contributes significantly to the regulation of plant defences and, as such, is a target for pathogen effectors. Bacterial effectors delivered by the type III and IV secretion systems have been shown to interact with components of the host UPS. Some of these effectors possess functional domains that are conserved in UPS enzymes, whilst others contain novel domains with ubiquitination activities. Relatively little is known about effector activities in eukaryotic microbial plant pathogens. Nevertheless, effectors from oomycetes that contain an RXLR motif for translocation to the inside of plant cells have been shown to suppress host defences. Annotation of the genome of one such oomycete, the potato late blight pathogen Phytophthora infestans, and protein-protein interaction assays to discover host proteins targeted by the RXLR effector AVR3a, have revealed that this eukaryotic plant pathogen also has the potential to manipulate host plant UPS functions.

Runaway repeats force expansion of the Phytophthora infestans genome

Sequencing of the potato late blight pathogen Phytophthora infestans provides insight into the structure and evolution of its genome.

Gene expression changes during asexual sporulation by the late blight agent Phytophthora infestans occur in discrete temporal stages

Transcriptional changes during asexual sporangia formation by the late blight pathogen Phytophthora infestans were identified using microarrays representing 15,646 genes and RNA from sporulation time-courses, purified spores, and sporulation-defective strains. Results were confirmed by reverse transcription-polymerase chain reaction analyses of sporulation on artificial media and infected tomato. During sporulation, about 12% of genes were up-regulated compared to vegetative hyphae and 5% were down-regulated. The most prevalent induced genes had functions in signal transduction, flagella assembly, cellular organization, metabolism, and molecular or vesicular transport. Distinct patterns of expression were discerned based on the kinetics of mRNA induction and their persistence in sporangia. For example, most flagella-associated transcripts were induced very early in sporulation and maintained in sporangia, while many participants in metabolism or small molecule transport were also induced early but had low levels in sporangia. Data from this study are a resource for understanding sporogenesis, which is critical to the pathogenic success of P. infestans and other oomycetes.

A putative DEAD-box RNA-helicase is required for normal zoospore development in the late blight pathogen Phytophthorainfestans

The asexual multinucleated sporangia of Phytophthora infestans can germinate directly through a germ tube or indirectly by releasing zoospores. The molecular mechanisms controlling sporangial cytokinesis or sporangial cleavage, and zoospore release are largely unknown. Sporangial cleavage is initiated by a cold shock that eventually compartmentalizes single nuclei within each zoospore. Comparison of EST representation in different cDNA libraries revealed a putative ATP-dependent DEAD-box RNA-helicase gene in P. infestans, Pi-RNH1, which has a 140-fold increased expression level in young zoospores compared to uncleaved sporangia. RNA interference was employed to determine the role of Pi-RNH1 in zoospore development. Silencing efficiencies of up to 99% were achieved in some transiently-silenced lines. These Pi-RNH1-silenced lines produced large aberrant zoospores that had undergone partial cleavage and often had multiple flagella on their surface. Transmission electron microscopy revealed that cytoplasmic vesicles fused in the silenced lines, resulting in the formation of large vesicles. The Pi-RNH1-silenced zoospores were also sensitive to osmotic pressure and often ruptured upon release from the sporangia. These findings indicate that Pi-RNH1 has a major function in zoospore development and its potential role in cytokinesis is discussed.

Annual Meeting, Saskatoon, Saskatchewan, 2007 / Réunion annuelle, Saskatoon, Saskatchewan, 2007

Annual Meeting, Saskatoon, Saskatchewan, 2007 / Réunion annuelle, Saskatoon, Saskatchewan, 2007

Suppression of Cdc27B expression induces plant defence responses

Non-host resistance is the most general form of disease resistance in plants because it is effective against most phytopathogens. The importance of hypersensitive responses (HRs) in non-host resistance of Nicotiana species to the oomycete Phytophthora is clear. INF1 elicitin, an elicitor obtained from the late-blight pathogen Phytophthora infestans, is sufficient to induce a typical HR in Nicotiana species. The molecular mechanisms that underlie the non-host resistance component of plant defence responses have been investigated using differential-display polymerase chain reaction (PCR) in a model HR system between INF1 elicitin and tobacco BY-2 cells. Differential-display PCR has revealed that Cdc27B is down-regulated in tobacco BY-2 cells after treatment with INF1 elicitin. Cdc27B is one of 13 essential components of the anaphase-promoting complex or cyclosome (APC/C)-type E3 ubiquitin ligase complex in yeast. This APC/C-type E3 ubiquitin ligase complex regulates G2-to-M phase transition of the cell cycle by proteolytic degradation. In this study, we investigated the roles of this gene, NbCdc27B, in plant defence responses using virus-induced gene silencing. Suppression of NbCdc27B in Nicotiana benthamiana plants induced defence responses and a gain of resistance to Colletotrichum lagenarium fungus. Elicitin-induced hypersensitive cell death (HCD) was inhibited mildly in plants silenced with tobacco rattle virus::Cdc27B. Cdc27B could manage the signalling pathways of plant defence responses as a negative regulator without HCD.