Abstract:Phytophthora diseases cause widespread economic and environmental losses worldwide. Thousands of plant species are susceptible. Disease is typically initiated through the activity of motile, biflagellate zoospores. Plant penetration and colonisation are achieved through the secretion of a diverse range of cell wall-degrading enzymes and effector proteins. Effector proteins are especially important during biotrophic growth; they function to suppress host defence and regulate host metabolism to favour pathogen growth. Plants can detect the presence of Phytophthora cells and rapidly mount a basal defence response that often successfully inhibits disease development. A key aspect of basal defence is the formation of wall appositions that constitute a physical and chemical barrier to pathogen growth. Components of basal defence can be triggered by chemical and physical signals produced by invading Phytophthora cells. If basal defence fails to inhibit pathogen ingress, the plant cell under attack can undergo hypersensitive cell death. In Phytophthora–plant interactions, hypersensitive cell death can be triggered by elicitors or effectors. In the continuing arms race between pathogen and plant host, Phytophthora species have evolved a range of counter-defence mechanisms that include suppression of hypersensitive cell death, inhibition of plant degradative enzymes and protection against reactive oxygen species. This brief article provides an overview of the contribution of modern molecular cytology to our understanding of Phytophthora–plant interactions.
Abstract: Efficient nutrient acquisition is critical to the fitness of plant pathogens. To address how the late blight agent Phytophthora infestans adapts to nutrients offered by its hosts, genes in glycolytic, gluconeogenic and amino acid pathways were mined from its genome and their expression in different plant tissues and artificial media was measured. Evidence for conventional glycolytic and gluconeogenic processes was obtained, although several steps involved pyrophosphate-linked transformations which are uncommon in eukaryotes. In media manipulation studies, nearly all genes in the pathways were subject to strong transcriptional control. However in rye–sucrose media, tomato leaflets, potato tubers and, at both early and late stages of infection, most glycolytic genes were expressed similarly, which indicated that each plant tissue presented a nutrient-rich environment. Biochemical analyses also demonstrated that sporulation occurred from host material in which sugars were abundant, with fructose and glucose increasing at the expense of sucrose late in the disease cycle. The expression of only a few genes changed late in infection, with the most notable example being lower invertase levels in the sucrose-reduced leaves. Interestingly, most gluconeogenic genes were up-regulated in tubers compared with other tissues. Rather than reflecting a starvation response, this probably reveals the role of such enzymes in converting carbon skeletons from the abundant free amino acids of tubers into citric acid cycle and glycolysis intermediates, as genes involved in amino acid catabolism were also more highly expressed in tubers. The corresponding enzymes also displayed higher activities in defined media when amino acids were abundant, as in tubers.
Abstract: The potential to improve strawberry cultivation was assessed regarding the use the rolC genes from Agrobacterium rhizogenes that can confer
higher levels of free cytokinins. Strawberry (cv. Calypso) rolC lines were produced by genetic transformation of Agrobacterium tumefaciens. Yield and fruit quality of the control and transgenic lines were measured under open-field conditions. The effects of the transgenic rolC lines depended on gene copy number: rolC lines with one (Line A) or two gene (Line B) copies showed 30% greater yields than controls, due to 20% more fruit per plant and an increased fruit weight. Line A also differed in terms of the highest fruit quality, due to 10.5% increased soluble solids and 12.7% higher acidity. Moreover, cv. Calypso rolC lines A and B had increased tolerance to greenhouse infection by Phytophthora cactorum. Conversely, for all of these characters, Line F (five rolC copies) was not significantly different from the control line. The same lines were also used to examine their symbiosis with root arbuscular mycorrhizal fungi (AMF) using vital and non-vital staining of roots collected at different stages of plant growth. Control and rolC plants showed similar intensities of AMF infection according to plant phenology and/or physiology. Furthermore, possible horizontal gene transfer of the rolC gene was tested for the AMF spores by PCR, with all AMF samples negative using rolC primers. The use of the
rolC gene should be considered for the improvements provided in productivity, fruit quality and disease resistance of cultivated strawberry that show no effects on soil microorganisms.
Abstract: Oxylipins constitute a class of molecules notably involved in hostepathogen interactions. In the potato-Phytophthora infestans (Mont.) De Barry (P. infestans) relationships, the role of colneleic and colnelenic acids, two oxylipins resulting from the consecutive action of lipoxygenase
(EC 1.13.11.12) and divinyl ether synthase (EC 1.-) on respectively linoleic and linolenic acids have been previously reported. In the present paper, five potato cultivars with contrasting resistance to P. infestans were submitted to infection. Lipoxygenase pathway response was studied at both transcriptional and metabolic levels. A Northern blot preliminary study revealed that lipoxygenase (lox1 and lox3) and divinyl ether synthase genes were clearly up-regulated 96 h after leaf inoculation with P. infestans. Profiling of free and esterified oxylipins performed 24 h, 48 h, 72 h and 96 h after inoculation, showed that esterified oxylipins are mainly produced with 9-derivatives in higher concentrations (esterified forms of colnelenic acid, 9-hydroxy octadecatrienoic acid, 9-hydroperoxy octadecatrienoic acid). Oxylipin accumulation is undetectable 24 h after infection, slightly detectable after 48 h, reaching highest concentrations after 96 h. Cultivars show slightly different oxylipin profiles but the concentration of individual oxylipins differs markedly 96 h after infection. No correlation was found between P. infestans resistance levels and oxylipin synthesis rates or concentration. To assess local and systemic effects of colneleic acid application before P. infestans infection, Bintje cultivar was sprayed with colneleic acid 72 h before inoculation. Both application modes (local and systemic) resulted in lipoxygenase pathway activation without affecting the resistance level to the pathogen.
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 activation 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 identification 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 trafficking 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: Recent research has shown that cyclophilins, proteins that catalyze the isomerization of peptidyl–prolyl bonds, play a variety of important roles in infection, including facilitating host penetration and colonization and acti-
vating pathogen effector proteins within the host cytoplasm. In the current study, bioinformatic analysis of the genomes of three species of plant pathogens in the genus Phytophthora has revealed extensive synteny between the 20 or 21 members of the cyclophilin gene family. In P. infestans, extensive EST studies give evidence of the expression of 14 of the 21 genes. Sequences homologous to 12 of the 14 expressed P. infestans cyclophilins were isolated using PCR and gene-specific primers in the broad host range pathogen, P. nicotianae. Quantitative real-time PCR measurements of transcript levels in P. nicotianae at four stages of asexual development and during infection of resistant and susceptible tobacco plants gave evidence of expression of seven of the P. nicotianae homologs. The most abundantly expressed gene, PnCyPA, has a lower mRNA level in zoospores compared to other stages of asexual development and its expression increases during infection of susceptible plants. Immunocytochemical studies indicate that PnCyPA occurs in the nucleus and cytoplasm of P. nicotianae cells and is secreted from germinated cysts.
Abstract: The effect of infection by Phytophthora sojae on the oxidative state in the soybean cultivars Nannong 493-1 (medium-resistant) and Hefeng 35 (susceptible) was studied. Pro-oxidant activity (H2O2 production and lipid peroxidation) and antioxidant systems (enzymatic activities of catalase and glutathione reductase, and low-molecular-weight antioxidants such as reduced ascorbate and glutathione) along with cell death and pathogenesis-related (PR) protein analyses were performed to examine their roles in establishing resistance or susceptibility. During pathogen infection, H2O2 accumulation was higher in the soybean cultivar Nannong 493-1 than in Hefeng 35. Cell death was more severe in Nannong 493-1 during the early infection period. However, the malondialdehyde levels were higher in Hefeng 35 than in Nannong 493-1 in the late experiment period. The inherent levels of the antioxidant systems were not consistently higher or lower in Nannong 493-1 than in Hefeng 35 under different pathogen infection duration or antioxidation parameters. The expression of the PR genes induced by pathogen infection varied with the cultivar and the pathogen-infection process. Exogenous treatment of soybean with one of three reductants (dithiothreitol, reduced ascorbate, or glutathione) facilitated the disease caused by P. sojae in both cultivars. In general, these results show that reactive oxygen species play an important role in the interactions of soybean and P. sojae.
Abstract: Transgenic Phytophthora sojae strains that produce green fluorescent protein (GFP) were obtained after stable DNA integration using the Hsp70 promoter and the Ham34 terminator of Bremia lactucae. The expression of GFP during different developmental stages of P. sojae was observed using fluorescent microscopy. Based on this reporter system, the histopathologic events caused by the pathogen in soybean leaves, hypocotyls and roots were monitored. Meanwhile, the difference in resistance between different soybean cultivars against P. sojae was analyzed microscopically in roots. The results indicate that GFP can be stably expressed in zoosporangia, zoospores, cysts, hyphae and oospores of P. sojae. Using the GFP marker, the infecting pathogens in leaves, hypocotyls and roots of host could be distinctly visualized. The germ tube length of cysts germinating on the roots of resistant cultivar Nannong 8848 was longer than that on the roots of susceptible cultivar Hefeng 35. These results show for the first time that this eukaryotic reporter can be used in P. sojae as a stable and vital marker, allowing the study of genetics of this hemibiotrophic pathogen.
Abstract: Lateral gene transfer (LGT) can facilitate the acquisition of new functions in recipient lineages, which may enable them to colonize new environments. Several recent publications have shown that gene transfer between prokaryotes and eukaryotes occurs with appreciable frequency. Here we present a study of interdomain gene transfer of cutinases – well documented virulence factors in fungi – between eukaryotic plant pathogens Phytophthora species and prokaryotic bacterial lineages. Two putative cutinase genes were cloned from Phytophthora brassicae and Northern blotting experiments showed that these genes are expressed early during the infection of the host Arabidopsis thaliana and induced during cyst germination of the pathogen. Analysis of the gene organisation of this gene family in Phytophthora ramorum and P. sojae showed three and ten copies in tight succession within a region of 5 and 25 kb, respectively, probably indicating a recent expansion in Phytophthora lineages by gene duplications. Bioinformatic analyses identified orthologues only in three genera of Actinobacteria, and in two distantly related eukaryotic groups: oomycetes and fungi. Together with phylogenetic analyses this limited distribution of the gene in the tree of life strongly support a scenario where cutinase genes originated after the origin of land plants in a microbial lineage living in proximity of plants and subsequently were transferred between distantly related plant-degrading microbes. More precisely, a cutinase gene was likely acquired by an ancestor of P. brassicae, P. sojae, P. infestans and P. ramorum, possibly from an actinobacterial source, suggesting that gene transfer might be an important mechanism in the evolution of their virulence. These findings could indeed provide an interesting model system to study acquisition of virulence factors in these important plant pathogens.
Abstract: To explore the molecular mechanisms that are involved in the pathogenicity of Phytophthora sojae, a suppression subtractive hybridization method was developed to screen for P. sojae genes that are differentially expressed in the early stage of Glycine max (soybean) infection. A cDNA library enriched for upregulated parasite genes was generated; of the 73 genes that were found to be upregulated, 66 are significantly similar to sequences in the P. sojae genome, and seven have no significant similarities in the databases examined. These sequences are predicted to encode proteins involved in protein biosynthesis, energy production, cell signaling, cell-wall biogenesis, and transcription regulation. Virtual Northern assay of random selected seven genes revealed that they are all highly expressed in plant infection. Reverse transcriptase polymerase chain reaction was used to further examine the expression pattern of these genes during soybean infection. These results provide an important insight into the genes expressed during P. sojae infection of soybean, which may be involved in oomycete pathogenesis.
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