Late Blight Pathogen Phytophthora Infestans Research Articles

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.

Influence of elevated CO 2 and ozone concentrations on late blight resistance and growth of potato plants

Potato plants ( Solanum tuberosum L. cv. Indira) with high susceptibility to the late blight pathogen Phytophthora infestans were exposed for 4 weeks to two different CO 2 concentrations (400/700 ppm) combined with ambient and double ambient ozone concentrations (first experiment) and with 1/5 ambient and ambient ozone concentrations (second experiment) in climate chambers. Leaves of the potato plants were then inoculated with Phytophthora infestans zoospores. Plants from the “high CO 2” variant showed a significantly increased resistance to the pathogen, verified by visual evaluation and quantitative real-time PCR, whereas plants treated with double ambient ozone were slightly more susceptible. An increase in the constitutive activities of the PR-proteins β-1,3-glucanase and osmotin in leaves of plants exposed to 700 ppm CO 2 correlated with the increase in resistance at this CO 2-concentration. Biomass parameters were barely affected by the elevated CO 2-concentration but decreased with increasing ozone concentrations. Biochemical analyses revealed that the content of starch as well as the content of soluble sugars in leaves were highest at the double ambient ozone/700 ppm CO 2 variants pointing to an ozone-induced inhibition of assimilate allocation from leaves to tubers. Leaf C/N-ratio increased at elevated CO 2-concentrations due to a decrease in N-content. The effect of the ozone- and CO 2-induced biochemical changes on the resistance response of potato towards Phytophthora infestans is discussed.

Assessing resistance types and levels to epidemic diseases from the analysis of disease progress curves: Principles and application to potato late blight

Both race-specific (RS) and race-non-specific (RNS) resistances exist in potato against the late blight pathogenPhytophthora infestans. Because these resistance types do not have the same epidemiological effects, their presence, alone or combined, in potato genotypes can be deduced from the analysis of disease progress curves from field experiments, a type of data commonly available to potato breeders, and their comparison with those of standard reference cultivars. The identification of RS resistance is based on the presence of a delay in epidemic onset compared to a susceptible cultivar, whereas the identification of RNS resistance is translated into a reduction of apparent infection rates. These parameters can be easily computed after linearization of the disease progress curves. This paper assesses the reliability of this identification using sets of experimental data, discusses its limitations, and highlights potential applications for breeding and cultivar assessment purposes.

Effector-triggered immunity by the plant pathogen Phytophthora

A new genetic locus mediating avirulence in the potato late blight pathogen Phytophthora infestans has been discovered. The Avr3b-Avr10-Avr11 locus is recognized by three different potato resistance genes, and is different from other Avr loci that have been identified thus far. This locus encodes a large protein with a WD40 domain and sequence similarities to transcription factors. Multiple, truncated copies of this gene have arisen by gene amplification and are characteristic of avirulent strains of P. infestans. Here, we describe the new avirulence locus and discuss the importance of this finding.

Amplification generates modular diversity at an avirulence locus in the pathogen Phytophthora

The destructive late blight pathogen Phytophthora infestans is notorious for its rapid adaptation to circumvent detection mediated by plant resistance (R) genes. We performed comparative genomic hybridization on microarrays (array-CGH) in a near genome-wide survey to identify genome rearrangements related to changes in virulence. Six loci with copy number variation were found, one of which involves an amplification colocalizing with a previously identified locus that confers avirulence in combination with either R gene R3b, R10, or R11. Besides array-CGH, we used three independent approaches to find candidate genes at the Avr3b-Avr10-Avr11 locus: positional cloning, cDNA-AFLP analysis, and Affymetrix array expression profiling. This resulted in one candidate, pi3.4, that encodes a protein of 1956 amino acids with regulatory domains characteristic for transcription factors. Amplification is restricted to the 3' end of the full-length gene but the amplified copies still contain the hallmarks of a regulatory protein. Sequence comparison showed that the amplification may generate modular diversity and assist in the assembly of novel full-length genes via unequal crossing-over. Analyses of P. infestans field isolates revealed that the pi3.4 amplification correlates with avirulence; isolates virulent on R3b, R10, and R11 plants lack the amplified gene cluster. The ancestral state of 3.4 in the Phytophthora lineage is a full-length, single-copy gene. In P. infestans, however, pi3.4 is a dynamic gene that is amplified and has moved to other locations. Modular diversity could be a novel mechanism for pathogens to quickly adapt to changes in the environment.

Peroxynitrite Generation and Tyrosine Nitration in Defense Responses in Tobacco BY-2 Cells

Peroxynitrite (ONOO(-)) is a compound formed by reaction of superoxide (O(2) (-)) with nitric oxide (NO) and is expected to possess characteristics of both O(2) (-) reactivity and NO mobility in order to function as a signal molecule. Although there are several reports that describe the role of ONOO(-) in defense responses in plants, it has been very difficult to detect ONOO(-) in bioimaging due to its short half-life or paucity of methods for ONOO(-)-specific detection among reactive oxygen species or free radicals. Aminophenyl fluorescein (APF), a recently developed novel fluorophore for direct detection of ONOO(-) in bioimaging, was used for intracellular ONOO(-) detection. ONOO(-) generation in tobacco BY-2 cells treated with INF1, the major elicitin secreted by the late blight pathogen Phytophthora infestans, occurred within 1 h and reached a maximum level at 6-12 h after INF1 treatment. Urate, a ONOO(-) scavenger, abolished INF1-induced ONOO(-) generation. It is well known that ONOO(-) reacts with tyrosine residues in proteins to form nitrotyrosine in a nitration reaction as an ONOO(-)-specific reaction. Western blot analysis using anti-nitrotyrosine antibodies recognized nitrotyrosine-containing proteins in 20 and 50 kDa bands in BY-2 protein extract containing SIN-1 [3-(4-morpholinyl) sydnonimine hydrochloride; an ONOO(-) donor]. These bands were also recognized in INF1-treated BY-2 cells and were found to be slightly suppressed by urate. Our study is the first to report ONOO(-) detection and tyrosine nitration in defense responses in plants.

Influence of compost tea, powdered kelp, and Manzate® 75 on bacterial-community composition, and antibiosis against Phytophthora infestans in the potato phylloplane

The search for replacements for synthetic fungicides has prompted the examination of a range of plant-based composts and extracts as alternative crop-protection agents. A study was established to compare the relative efficacy of the commercially available Jolly Farmer (JF) compost tea, of an Acadian SeaPlants Inc. (ASL) powdered-kelp product, and of Manzate® 75 DF (dry flowable) as foliar protectants against the potato late blight pathogen Phytophthora infestans. Population density, community structure, and antibiosis ability of phylloplane bacteria present on potato foliage ('Russet Burbank') were compared before and after foliar treatment. Bacteria recovered using culture-dependent means were characterized by fatty acid methyl ester (FAME) analysis and partial sequencing of their 16S ribosomal RNA genes. Autochthonous bacterial-population densities on the phylloplane varied with year, and tended to be lowest following Manzate 75 DF treatments. Phyllobacteria communities recovered from posttreatment phylloplanes often bore little resemblance to their tank-mixture counterparts, for all three treatments, and were significantly (P=0.05) less effective at inhibiting P. infestans growth, in vitro, compared with foliar communities before the treatments. Manzate 75 DF was the most effective treatment (P=0.05) for controlling P. infestans infection and disease development in vivo. Both JF compost tea and ASL powdered kelp were similar in their inability to protect potato leaves from P. infestans severity. Phyllobacterial populations established following Manzate 75 DF treatment proved to have significantly (P=0.01) fewer antibiotic-producing strains (in vitro) than naturally occurring leaf populations prior to foliar treatment. Accordingly, it appears that Manzate 75 DF derives little if any additive or synergistic plant-protectant properties from the naturally established phylloflora.

Susceptibility of five nightshade (Solanum) species tophytophthora infestans

The susceptibility of leaves and berries of five night-shade species to infection by the potato late blight pathogenPhytophthora infestans was tested. American black nightshade (Solanum americanum) and eastern black nightshade (Solanum ptycanthum) were resistant to infection. However, two accessions (entire-edged and dentate-edged leaf) of hairy nightshade (Solanum sarrachoides), cutleaf nightshade (Solanum triflorum), and bitter or climbing nightshade (Solanum dulcamara) were susceptible. Survival of the pathogen in susceptible species was tested under different environmental conditions. The pathogen survived for at least 2 wk in both hairy nightshade accessions when plant material was kept in soil at a temperature of 20 C. However,P. infestans survived 48 h at −15 C only in the entire-leaf hairy nightshade accession. In the laboratory, oospores of the pathogen were formed in inoculated plants of the two hairy nightshade accessions, cutleaf nightshade, and ‘Russet Burbank’ potato, but not in American black nightshade, bitter nightshade, or eastern black nightshade.

Comparative genomics enabled the isolation of the R3a late blight resistance gene in potato

Comparative genomics provides a tool to utilize the exponentially increasing sequence information from model plants to clone agronomically important genes from less studied crop species. Plant disease resistance (R) loci frequently lack synteny between related species of cereals and crucifers but appear to be positionally well conserved in the Solanaceae. In this report, we adopted a local RGA approach using genomic information from the model Solanaceous plant tomato to isolate R3a, a potato gene that confers race-specific resistance to the late blight pathogen Phytophthora infestans. R3a is a member of the R3 complex locus on chromosome 11. Comparative analyses of the R3 complex locus with the corresponding I2 complex locus in tomato suggest that this is an ancient locus involved in plant innate immunity against oomycete and fungal pathogens. However, the R3 complex locus has evolved after divergence from tomato and the locus has experienced a significant expansion in potato without disruption of the flanking colinearity. This expansion has resulted in an increase in the number of R genes and in functional diversification, which has probably been driven by the co-evolutionary history between P. infestans and its host potato. Constitutive expression was observed for the R3a gene, as well as some of its paralogues whose functions remain unknown.

Dissection of foliage and tuber late blight resistance in mapping populations of potato

The devastating late blight pathogen Phytophthora infestans infects foliage as well as tubers of potato. To date, resistance breeding has often focused on foliage blight resistance, but tuber blight resistance is becoming more and more important in cultivated potatoes. In this study, a reliable tuber assay for resistance assessment was developed and foliage and tuber blight resistance (R) was compared in four mapping populations. In the RH4X-103 population, tuber blight resistance inherited independently from foliage blight resistance. Three specific R genes against P. infestans were segregating. The Rpi-abpt and R3a genes function as foliage-specific R genes, whereas the R1 gene acts on both foliage and tuber. In the segregating populations SHRH and RH94-076, tuber and foliage blight resistance correlated significantly, which suggests that resistance in foliage and tuber is conferred by the same gene (could be R3b) and quantitative trait loci (QTL), respectively. In the CE population neither tuber nor foliage resistance was observed.

Prosystemin-antimicrobial-peptide fusion reduces tomato late blight lesion expansion

Antimicrobial peptides offer a new method for controlling pathogens, however, many promising peptides are too small for direct production in plants. A protein delivery system was developed based on a proteolytic mechanism used by Solanaceous plants to produce the very small (18 amino acid) signaling peptide systemin from the polypeptide prosystemin. Fusion of the gene encoding the 23 kDa protein prosystemin with the antimicrobial peptide (pep11) sequence, replacing the systemin sequence, allowed for expression in transgenic tomato plants. Six days after inoculation with the late blight pathogen Phytophthora infestans, detached leaflets of transgenic tomato (Rutgers) exhibited a reduction in lesion size of at least 50 percent.

Origins, lineage-specific expansions, and multiple losses of tyrosine kinases in eukaryotes.

Tyrosine kinases are important components of metazoan signaling pathways, and their mutant forms are implicated in various malignancies. Searching the sequences from the genomes of 28 eukaryotes and the GenBank, we found tyrosine kinases not only in metazoans but also in the green algae Chlamydomonas reinhardtii, the potato late blight pathogen Phytophthora infestans, and the protozoan pathogen Entamoeba histolytica, contrary to the current view that tyrosine kinases are animal-specific. Based on a phylogenetic analysis, we divided this gene family into 43 subfamilies and found that at least 19 tyrosine kinases were likely present in the common ancestor of chordates, arthropods, and nematodes. Interestingly, most of the subfamilies have conserved domain organizations among subfamily members but have undergone different degrees of expansion during the evolution of metazoans. In particular, a large number of duplications occurred in the lineage leading to the common ancestor of Tagifugu and mammals after its split from the Ciona lineage about 450 to 550 MYA. The timing of expansion coincides with proposed large-scale duplication event in the chordate lineage. Furthermore, gene losses have occurred in most subfamilies. Interestingly, different subfamilies have similar net gain rates in the chordates studied. However, the tyrosine kinases in mouse and human or in fruit fly and mosquito mostly have a one-to-one relationship between species, indicating that static periods of 90 Myr or longer in tyrosine kinase evolution have followed large expansion events.

Involvement of nitric oxide generation in hypersensitive cell death induced by elicitin in tobacco cell suspension culture

Recent studies suggest that nitric oxide (NO), an important signaling and defense molecule in mammals, plays a key role in activating disease resistance in plants. We characterized NO production by tobacco Bright Yellow-2 cells pharmacologically after treatment with INF1, the major elicitin secreted by the late blight pathogen Phytophthora infestans, prepared from Escherichia coli. NO production rapidly occurred within 1 h and reached a maximum level 3–6 h after the addition of INF1. Carboxy-PTIO, a NO-specific scavenger, abolished INF1-induced NO production in a dose-dependent manner. Pretreatment of protein synthesis inhibitor cycloheximide and protein kinase inhibitor K252a blocked NO production 3–12 h after INF1 treatment, indicating that NO production requires de novo protein synthesis and protein phosphorylation. In an investigation of the relations between NO generation and several defense responses induced by INF1, carboxy-PTIO completely suppressed activation of a 41-kDa protein kinase and cell death by INF1. Carboxy-PTIO also suppressed the induction of hypersensitive-related (hsr) genes HSR515 and HSR203J, the expression of which is strongly correlated with the hypersensitive response in plants. The results suggest that NO plays a crucial role in the induction of hypersensitive cell death.

A Galpha subunit controls zoospore motility and virulence in the potato late blight pathogen Phytophthora infestans.

The heterotrimeric G-protein pathway is a ubiquitous eukaryotic signalling module that is known to regulate growth and differentiation in many plant pathogens. We previously identified Pigpa1, a gene encoding a G-protein alpha subunit from the potato late blight pathogen Phytophthora infestans. P. infestans belongs to the class oomycetes, a group of organisms in which signal transduction processes have not yet been studied at the molecular level. To elucidate the function of Pigpa1, PiGPA1-deficient mutants were obtained by homology-dependent gene silencing. The Pigpa1-silenced mutants produced zoospores that turned six to eight times more frequently, causing them to swim only short distances compared with wild type. Attraction to the surface, a phenomenon known as negative geotaxis, was impaired in the mutant zoospores, as well as autoaggregation and chemotaxis towards glutamic and aspartic acid. Zoospore production was reduced by 20-45% in different Pigpa1-silenced mutants. Transformants expressing constitutively active forms of PiGPA1, containing amino acid substitutions (R177H and Q203L), showed no obvious phenotypic differences from the wild-type strain. Infection efficiencies on potato leaves ranged from 3% to 14% in the Pigpa1-silenced mutants, compared with 77% in wild type, showing that virulence is severely impaired. The results prove that PiGPA1 is crucial for zoospore motility and for pathogenicity in an important oomycete plant pathogen.

Phospholipase D in Phytophthora infestans and its role in zoospore encystment.

We show that differentiation of zoospores of the late blight pathogen Phytophthora infestans into cysts, a process called encystment, was triggered by both phosphatidic acid (PA) and the G-protein activator mastoparan. Mastoparan induced the accumulation of PA, indicating that encystment by mastoparan most likely acts through PA. Likewise, mechanical agitation of zoospores, which often is used to induce synchronized encystment, resulted in increased levels of PA. The levels of diacylglycerolpyrophosphate (DGPP), the phosphorylation product of PA, increased simultaneously. Also in cysts, sporangiospores, and mycelium, mastoparan induced increases in the levels of PA and DGPP. Using an in vivo assay for phospholipase D (PLD) activity, it was shown that the mastoparan-induced increase in PA was due to a stimulation of the activity of this enzyme. Phospholipase C in combination with diacylglycerol (DAG) kinase activity also can generate PA, but activation of these enzymes by mastoparan was not detected under conditions selected to highlight 32P-PA production via DAG kinase. Primary and secondary butanol, which, like mastoparan, have been reported to activate G-proteins, also stimulated PLD activity, whereas the inactive tertiary isomer did not. Similarly, encystment was induced by n- and sec-butanol but not by tert-butanol. Together, these results show that Phytophthora infestans contains a mastoparan- and butanol-inducible PLD pathway and strongly indicate that PLD is involved in zoospore encystment. The role of G-proteins in this process is discussed.

Primary structure and tissue‐specific expression of the pathogenesis‐related protein PR‐1b in potato†

Summary The infection of potato (Solanum tuberosum) leaves with the late blight pathogen Phytophthora infestans, or treatment with fungal elicitor, leads to the massive accumulation of pathogenesis-related (PR) proteins in the extracellular leaf space. The most abundant of these proteins was purified to apparent homogeneity and identified as a new, basic member of the PR-1 family of defence proteins, designated PR-1b. Antibodies raised against the protein and a cDNA isolated by differential screening were used to study the temporal and spatial patterns of PR-1b protein and mRNA distribution in healthy and infected potato tissues. PR-1b was present in old leaves and at low levels also in the carpels of flowers. In leaves, strong accumulation of PR-1b mRNA and protein occurred in response to infection by the oomycete pathogen Phytophthora infestans or the bacterial pathogen Pseudomonas syringae pv. maculicola. PR-1b mRNA and protein accumulation was clearly initiated at the infection site, but a delayed and sustained accumulation was also observed in neighbouring, uninfected leaves of potato plants. Tissue- and cell type-specific expression of PR-1b was analysed by immunohistochemical and in situ RNA hybridization techniques. Appreciable amounts of PR-1b protein and mRNA were localized in epidermal cells, guard cells of the stomata, glandular trichomes, crystal idioblasts, and cells of the vascular system of infected leaves. However, no significant differences in the amounts and distribution patterns of PR-1b could be observed between compatible and incompatible interactions of potato and Phytophthora infestans, indicating that PR-1b expression is not involved in determining cultivar/race-specific resistance in potato.

Increased ratio of mitochondrial rDNA to cytoplasmic rDNA during zoosporic and germinating cyst stages of the life cycle of Phytophthora infestans (Mont.) de Bary.

A differential RNA display approach was used to study the gene expression in zoospores (Z) and germinating cysts (GC) of the late blight pathogen Phytophthora infestans. Four differentially amplified cDNAs were selected and cloned. The clone pGPiZ0.5 showed a 2.7-kb transcript highly expressed in Z. A BLAST search revealed an almost full sequence homology (98%) to the P. infestans mitochondrial large subunit rRNA. Northern blot analysis showed a twofold accumulation of the mitochondrial rRNA (mit rRNA) in Z compared with that of GC and mycelia of P. infestans. The high level of mit rRNA in Z might reflect an increased number of gene copies, an increased rDNA transcription rate, or both. Dot blot experiments indicated that the amount of mitochondrial rDNA (mit rDNA) relative to cytoplasmic rDNA is twofold higher in Z and GC than in mycelia. This relatively elevated mit rDNA could explain the high level of mit rRNA in the zoosporic phase. On the contrary, GC conserves the mit rDNA content, but the level of mit rRNA drops to 50% that of Z. The data are consistent with a very active mitochondrial protein synthesis during zoosporic phase, followed by a rapid down-regulation of mitochondrial activity during cyst formation.

Oospore Germination and Formation by the Late Blight Pathogen Phytophthora infestans in vitro and under Field Conditions

The ability of the late blight pathogen Phytophthora infestans to form oospores in leaves of seven potato cultivars was examined at different incubation temperatures under controlled environmental conditions and under field conditions. At 10°C, the oospore formation in three intermediate‐resistant cultivars all differed significantly from each other (P < 0.05), with the lowest amount formed in cv. Asterix. This latter cultivar did not form oospores at any other temperature. Under field conditions oospores were formed abundantly in a naturally infected field. A significant date by cultivar interaction showed that P. infestans increased the oospore formation in foliage by time in cvs Columbo, Hertha and Matilda, whereas no significant differences between dates were found for other cultivars. The genetic structure of P. infestans in the naturally infected field plot, where oospores formed abundantly, was studied by using amplified fragment length polymorphism and a high genetic diversity was revealed. Oospore germination from two Scandinavian (A1 and A2) P. infestans isolates was stimulated in visible light and in 1 : 2 and 1 : 10 soil extract. The effect of light and nutrients on oosporogenesis is discussed.

Hydroperoxides of fatty acids induce programmed cell death in tomato protoplasts

Arachidonic acid (AA) is a fatty acid elicitor abundant in the glycerolipids of the late blight pathogen Phytophthora infestans and related Oomycete species. Lipoxygenases (LOX), which catalyze the addition of molecular oxygen to the 1 or 5 position of a cis, cis -1,4- Z, Z -pentadiene system in polyunsaturated fatty acids, is induced in host plants such as tomato and potato during infection by P. infestans. Here it is reported that AA, the LOX metabolites of AA, 5- and 15-hydroperoxyeicosatetraenioc acid (5- and 15-HpETE), and the LOX metabolite of linoleic acid, 9-hydroperoxyoctadecadienoic acid (9-HpODE), are potent inducers of programmed cell death (PCD) in tomato protoplasts. 5- and 15-HpETE increased DNA fragmentation as detected by t erminal deoxynucleotidyl transferase-mediated d U TP-X n ick e nd l abeling (TUNEL) and increased DNA laddering as visualized by ligation-mediated PCR. Background levels of DNA laddering were decreased in intensity by Zn2+ and increased by Ca2+, effects that are consistent with the reported action of these cations on PCD-associated endonucleases in other systems. H2O2, methyl jasmonate, and linoleic and linolenic acids were not toxic to tomato protoplasts at concentrations up to 350μM, and lipid peroxides (LD100=80μM) were far more potent inducers of death than free AA within this same concentration range. These results indicate the potential of fatty acid peroxides and LOX-related metabolism to engage an apoptotic type of PCD in higher plant cells.