Convergent evolution of immune receptors underpins distinct elicitin recognition in closely related Solanaceous plants.

Elicitins are a large family of secreted proteins in Phytophthora. Clade 1 elicitins were identified decades ago as potent elicitors of immune responses in Nicotiana species, but the mechanisms underlying elicitin recognition are largely unknown. Here we identified an elicitin receptor in Nicotiana benthamiana that we named REL for Responsive to ELicitins. REL is a receptor-like protein (RLP) with an extracellular leucine-rich repeat (LRR) domain that mediates Phytophthora resistance by binding elicitins. Silencing or knocking out REL in N. benthamiana abolished elicitin-triggered cell death and immune responses. Domain deletion and site-directed mutagenesis revealed that the island domain (ID) located within the LRR domain of REL is crucial for elicitin recognition. In addition, sequence polymorphism in the ID underpins the genetic diversity of REL homologs in various Nicotiana species in elicitin recognition and binding. Remarkably, REL is phylogenetically distant from the elicitin response (ELR) protein, an LRR-RLP that was previously identified in the wild potato species Solanum microdontum and REL and ELR differ in the way they bind and recognize elicitins. Our findings provide insights into the molecular basis of plant innate immunity and highlight a convergent evolution of immune receptors towards perceiving the same elicitor.

Antifungal activity of zedoary turmeric oil against Phytophthora capsici through damaging cell membrane.

Phytophthora capsici is a plant oomycete pathogen, which causes many devastating diseases on a broad range of hosts. Zedoary turmeric oil (ZTO) is a kind of natural plant essential oil that has been widely used in pharmaceutical applications. However, the antifungal activity of ZTO against phytopathogens remains unknown. In this study, we found ZTO could inhibit P. capsici growth and development in vitro and in detached cucumber and Nicotiana benthamiana leaves. Besides, ZTO treatment resulted in severe damage to the cell membrane of P. capsici, leading to the leakage of intracellular contents. ZTO also induced a significant increase in relative conductivity, malondialdehyde concentration and glycerol content. Furthermore, we identified 50 volatile organic compounds from ZTO, and uncovered Curcumol, ß-elemene, curdione and curcumenol with strong inhibitory activities against mycelial growth of P. capsici. Overall, our results not only shed new light on the antifungal mechanism of ZTO, but also imply a promising alternative for the control of phytophthora blight caused by P. capsici.

A conserved RxLR effector interacts with host RABA-type GTPases to inhibit vesicle-mediated secretion of antimicrobial proteins.

Plant pathogens of the oomycete genus Phytophthora produce virulence factors, known as RxLR effector proteins that are transferred into host cells to suppress disease resistance. Here, we analyse the function of the highly conserved RxLR24 effector of Phytophthora brassicae. RxLR24 was expressed early in the interaction with Arabidopsis plants and ectopic expression in the host enhanced leaf colonization and zoosporangia formation. Co-immunoprecipitation (Co-IP) experiments followed by mass spectrometry identified different members of the RABA GTPase family as putative RxLR24 targets. Physical interaction of RxLR24 or its homologue from the potato pathogen Phytophthora infestans with different RABA GTPases of Arabidopsis or potato, respectively, was confirmed by reciprocal Co-IP. In line with the function of RABA GTPases in vesicular secretion, RxLR24 co-localized with RABA1a to vesicles and the plasma membrane. The effect of RxLR24 on the secretory process was analysed with fusion constructs of secreted antimicrobial proteins with a pH-sensitive GFP tag. PATHOGENESIS RELATED PROTEIN 1 (PR-1) and DEFENSIN (PDF1.2) were efficiently exported in control tissue, whereas in the presence of RxLR24 they both accumulated in the endoplasmic reticulum. Together our results imply a virulence function of RxLR24 effectors as inhibitors of RABA GTPase-mediated vesicular secretion of antimicrobial PR-1, PDF1.2 and possibly other defence-related compounds.

Phenotypic analysis of Phytophthora parasitica by using high throughput phenotypic microarray.

OBJECTIVE: We studied the phenotypic characterization of Phytophthora parasitica Dastur var. nicotianae. METHODS: Phenotypic characterization of the pathogen was studied to provide information for disease management program by using BIOLOG phenotype MicroArray (PM ). Using PM plates 1 to 10, 950 different phenotypic characterizations were tested. RESULTS: P. parasitica was able to metabolize 74% of tested carbon sources, 96% of nitrogen sources, 100% of sulfur sources, and 98% of phosphorus sources. Most informative utilization patterns for carbon sources of P. parasitica were organic acids and carbohydrates, and for nitrogen were various amino acids. The pathogen presented 285 different nitrogen pathways. It had wide range adaptabilities in osmolytes with up to 1% sodium chloride, up to 3% potassium chloride, up to 5% sodium sulfate, up to 20% ethylene glycol, up to 2% sodium formate, up to 5% urea, and up to 2% sodium lactate. It also exhibited active metabolism under pH values between 3.5 and 10, with optimal pH of around 7.0. The pathogen showed both decarboxylase and deaminase activities in the presence of various amino acids. CONCLUSION: These phenotypic characterizations of P. parasitica provided the theoretical basis for the next study of the pathogen in physiology and metabolism, and provided potential new way for tobacco black shank management.

Comparative structural and functional analysis of genes encoding pectin methylesterases in Phytophthora spp.

We have scanned the Phytophthora infestans, P. ramorum, and P. sojae genomes for the presence of putative pectin methylesterase genes and conducted a sequence analysis of all gene models found. We also searched for potential regulatory motifs in the promoter region of the proposed P. infestans models, and investigated the gene expression levels throughout the course of P. infestans infection on potato plants, using in planta and detached leaf assays. We found that genes located on contiguous chromosomal regions contain similar motifs in the promoter region, indicating the possibility of a shared regulatory mechanism. Results of our investigations also suggest that, during the pathogenicity process, the expression levels of some of the analyzed genes vary considerably when compared to basal expression observed in in vitro cultures of non-sporulating mycelium. These results were observed both in planta and in detached leaf assays.

[Effects of elicitors on accumulation of phenolic acids and tanshinones in Salvia miltiorrhiza hairy root].

OBJECTIVE: To observe the effects of a biotic elicitor fungal hyphae extract, an abiotic elicitor methyl jasmonate and their synergistic action on the accumulation of phenolic acids and tanshinones in Salvia miltiorrhiza hairy root. METHOD: Different elicitors were added to S. miltiorrhiza hairy root, which was subcultured for 21 days, the dry weight and contents of phenolic acids and tanshinones were determined at different harvest-time. RESULT: S. miltiorrhiza hairy root growth was significantly inhibited by all three treatments and the accumulation of cryptotanshinone and dihydrotanshinone were promoted by each elicition. As for the accumulation of phenolic acids, there were differences between fungal elicitor and methyl jasmonate treatments, they were promoted by methyl jasmonate while inhibited in a certain extent by fungal hyphae extract. CONCLUSION: Fungal elicitor, methyl jasmonate and their synergistic action have significant influence on accumulation of components in S. miltiorrhiza hairy root, and the effect varies between phenolic acids and tanshinones. There is no correlation between production of water-soluble ingredients and fat-soluble components on the whole under three different treatments.

Alpha- and beta-tubulin from Phytophthora capsici KACC 40483: molecular cloning, biochemical characterization, and antimicrotubule screening.

Internal fragments of alpha- and beta-tubulin genes were generated using reverse transcription polymerase chain reaction (RT-PCR), and the termini were isolated using 5'- and 3'-rapid amplification of cDNA ends. Phytophthora capsici alpha- and beta-tubulin specific primers were then used to generate full-length cDNA by RT-PCR. The recombinant alpha- and beta-tubulin genes were expressed in Escherichia coli BL21 (DE3), purified under denaturing conditions, and average yields were 3.38-4.5 mg of alpha-tubulin and 2.89-4.0 mg of beta-tubulin, each from 1-l culture. Optimum conditions were obtained for formation of microtubule-like structures. A value of 0.12 mg/ml was obtained as the critical concentration of polymerization of P. capsici tubulin. Benomyl inhibited polymerization with half-maximal inhibition (IC(50)) = 468 +/- 20 microM. Approximately 18.66 +/- 0.13 cysteine residues per tubulin dimer were accessible to 5,5'-dithiobis-(2-nitrobenzoic acid), a quantification reagent of sulfhydryl and 12.43 +/- 0.12 residues were accessible in the presence of 200 microM benomyl. The order of preference for accessibility to cysteines was benomyl > colchicine > GTP > taxol, and cysteine accessibility changes conformed that binding sites of these ligands in tubulin were folding correctly. Fluorescence resonance energy transfer technique was used for high throughput screening of chemical library in search of antimitotic agent. There was significant difference in relative fluorescence by 210-O-2 and 210-O-14 as compared to colchicine.

Plasmodium falciparum and Hyaloperonospora parasitica effector translocation motifs are functional in Phytophthora infestans.

The oomycete potato late blight pathogen, Phytophthora infestans, and the apicomplexan malaria parasite Plasmodium falciparum translocate effector proteins inside host cells, presumably to the benefit of the pathogen or parasite. Many oomycete candidate secreted effector proteins possess a peptide domain with the core conserved motif, RxLR, located near the N-terminal secretion signal peptide. In the Ph. infestans effector Avr3a, RxLR and an additional EER motif are essential for translocation into host cells during infection. Avr3a is recognized in the host cytoplasm by the R3a resistance protein. We have exploited this cytoplasmic recognition to report on replacement of the RxLR-EER of Avr3a with the equivalent sequences from the intracellular effectors ATR1NdWsB and ATR13 from the related oomycete pathogen, Hyaloperonospora parasitica, and the host targeting signal from the Pl. falciparum virulence protein PfHRPII. Introduction of these chimeric transgenes into Ph. infestans and subsequent virulence testing on potato plants expressing R3a demonstrated the alternative motifs to be functional in translocating Avr3a inside plant cells. These results suggest common mechanisms for protein translocation in both malaria and oomycete pathosystems.

The Phytophthora infestans avirulence gene Avr4 encodes an RXLR-dEER effector.

Resistance in potato against the oomycete Phytophthora infestans is conditioned by resistance (R) genes that are introgressed from wild Solanum spp. into cultivated potato. According to the gene-for-gene model, proteins encoded by R genes recognize race-specific effectors resulting in a hypersensitive response (HR). We isolated P. infestans avirulence gene PiAvr4 using a combined approach of genetic mapping, transcriptional profiling, and bacterial artificial chromosome marker landing. PiAvr4 encodes a 287-amino-acid-protein that belongs to a superfamily of effectors sharing the putative host-cell-targeting motif RXLR-dEER. Transformation of P. infestans race 4 strains with PiAvr4 resulted in transformants that were avirulent on R4 potato plants, demonstrating that PiAvr4 is responsible for eliciting R4-mediated resistance. Moreover, expression of PiAvr4 in R4 plants using PVX agroinfection and agroinfiltration showed that PiAvr4 itself is the effector that elicits HR on R4 but not r0 plants. The presence of the RXLR-dEER motif suggested intracellular recognition of PiAvr4. This was confirmed in agroinfiltration assays but not with PVX agroinfection. Because there was always recognition of PiAvr4 retaining the signal peptide, extracellular recognition cannot be excluded. Deletion of the RXLR-dEER domain neither stimulated nor prevented elicitor activity of PiAvr4. Race 4 strains have frame shift mutations in PiAvr4 that result in truncated peptides; hence, PiAvr4 is apparently not crucial for virulence.

RXLR-mediated entry of Phytophthora sojae effector Avr1b into soybean cells does not require pathogen-encoded machinery.

Effector proteins secreted by oomycete and fungal pathogens have been inferred to enter host cells, where they interact with host resistance gene products. Using the effector protein Avr1b of Phytophthora sojae, an oomycete pathogen of soybean (Glycine max), we show that a pair of sequence motifs, RXLR and dEER, plus surrounding sequences, are both necessary and sufficient to deliver the protein into plant cells. Particle bombardment experiments demonstrate that these motifs function in the absence of the pathogen, indicating that no additional pathogen-encoded machinery is required for effector protein entry into host cells. Furthermore, fusion of the Avr1b RXLR-dEER domain to green fluorescent protein (GFP) allows GFP to enter soybean root cells autonomously. The conclusion that RXLR and dEER serve to transduce oomycete effectors into host cells indicates that the >370 RXLR-dEER-containing proteins encoded in the genome sequence of P. sojae are candidate effectors. We further show that the RXLR and dEER motifs can be replaced by the closely related erythrocyte targeting signals found in effector proteins of Plasmodium, the protozoan that causes malaria in humans. Mutational analysis of the RXLR motif shows that the required residues are very similar in the motifs of Plasmodium and Phytophthora. Thus, the machinery of the hosts (soybean and human) targeted by the effectors may be very ancient.

Cellulose synthesis in Phytophthora infestans is required for normal appressorium formation and successful infection of potato.

Cellulose, the important structural compound of cell walls, provides strength and rigidity to cells of numerous organisms. Here, we functionally characterize four cellulose synthase genes (CesA) in the oomycete plant pathogen Phytophthora infestans, the causal agent of potato (Solanum tuberosum) late blight. Three members of this new protein family contain Pleckstrin homology domains and form a distinct phylogenetic group most closely related to the cellulose synthases of cyanobacteria. Expression of all four genes is coordinately upregulated during pre- and early infection stages of potato. Inhibition of cellulose synthesis by 2,6-dichlorobenzonitrile leads to a dramatic reduction in the number of normal germ tubes with appressoria, severe disruption of the cell wall in the preinfection structures, and a complete loss of pathogenicity. Silencing of the entire gene family in P. infestans with RNA interference leads to a similar disruption of the cell wall surrounding appressoria and an inability to form typical functional appressoria. In addition, the cellulose content of the cell walls of the silenced lines is >50% lower than in the walls of the nonsilenced lines. Our data demonstrate that the isolated genes are involved in cellulose biosynthesis and that cellulose synthesis is essential for infection by P. infestans.

A translocation signal for delivery of oomycete effector proteins into host plant cells.

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 infestans RXLR-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 protein. We show that Avr3a, with or without 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 Avr3a RXLR-EER motifs with 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.

Characterization of a heat-stable protein with antimicrobial activity from Arabidopsis thaliana.

A heat-stable protein with antimicrobial activity was isolated from Arabidopsis thaliana plants by buffer-soluble extraction and two chromatographic procedures. The results of MALDI-TOF analysis revealed that the isolated protein shares high sequence identity with aspen SP1. To determine the exact antimicrobial properties of this protein, a cDNA encoding the protein was isolated from an A. thaliana leaf cDNA library and named AtHS1. AtHS1 mRNA was induced by exposure to external stresses, such as salicylic acid and jasmonic acid. We also analyzed the antimicrobial activity of recombinant AtHS1 expressed in Escherichia coli. This protein inhibited pathogenic fungal strains, except for Phytophthora infestans and Phytophthora nicotianae, and it exhibited antibacterial activity against E. coli and Staphylococcus aureus. These results suggest that AtHS1 shows good potential for use as a natural material in the study of antimicrobial agents.

Elevated amino acid biosynthesis in Phytophthora infestans during appressorium formation and potato infection.

Appressorium formation is believed to be an important event in establishing a successful interaction between the late blight pathogen, Phytophthora infestans, and its host plants potato and tomato. An understanding of molecular events occurring in appressorium development could suggest new strategies for controlling late blight. We used parallel studies of the transcriptome and proteome to identify genes and proteins that are up-regulated in germinating cysts developing appressoria. As a result, five distinct genes involved in amino acid biosynthesis were identified that show increased expression in germinating cysts with appressoria. These are a methionine synthase (Pi-met1), a ketol-acid reductoisomerase (Pi-kari1), a tryptophan synthase (Pi-trp1), an acetolactate synthase (Pi-als1), and a threonine synthase (Pi-ts1). Four of these P. infestans genes were also up-regulated, although to lower levels, during the early, biotrophic phase of the interaction in potato and all five were considerably up-regulated during the transition (48 hpi) to the necrotrophic phase of the interaction. Real-time RT-PCR revealed that expression of potato homologues of the amino acid biosynthesis genes increased during biotrophic and necrotrophic infection phases. Furthermore, we investigated levels of free amino acids in the pre-infection stages and found that in most cases there was a decrease in free amino acids in zoospores and germinating cysts, relative to sporangia, followed by a sharp increase in germinating cysts with appressoria. Amino acid biosynthesis would appear to be important for pathogenicity in P. infestans, providing a potential metabolic target for chemical control.

Specific formation of arachidonic acid and eicosapentaenoic acid by a front-end Delta5-desaturase from Phytophthora megasperma.

The biosynthesis of arachidonic acid (20:4(Delta5Z,8Z,11Z,14Z)) from linoleic acid in plants by transgenic means requires the sequential and specific action of two desaturation reactions and one elongation reaction. Here, we describe the isolation of a specific acyl-lipid-desaturase catalyzing the formation of the double bond at position 5 from a cDNA library from Phytophthora megasperma. The isolated full-length cDNA harbors a sequence of 1740 bp encoding a protein of 477 amino acids with a calculated molecular weight of 53.5 kDa. The desaturase sequence contained a predicted N-terminal cytochrome b(5)-like domain, as well as three histidine-rich domains. For functional identification, the cDNA was expressed in Saccharomyces cerevisiae, and the formation of newly formed fatty acids was analyzed. The expression of the heterologous enzyme resulted in the formation of arachidonic acid after di-homo-gamma-linolenic acid supplementation and in the formation of eicosapentaenoic acid synthesis from omega3-arachidonic acid. Results presented here on the substrate specificity identify this expressed protein as a classical Delta5-acyl-lipid-desaturase, capable of specifically introducing a double bond at the Delta5 position solely in 20-carbon-atom chain length fatty acids containing a double bond at position Delta8. Detailed analysis of the different lipid species showed a preferential occurrence of the desaturation reaction for fatty acids esterified to phosphatidylcholine.

Molecular cloning and biological activity of alpha-, beta-, and gamma-megaspermin, three elicitins secreted by Phytophthora megasperma H20.

We report on the molecular cloning of the Phytophthora megasperma H20 (PmH20) glycoprotein shown previously as an inducer of the hypersensitive response, of localized acquired resistance and of systemic acquired resistance in tobacco (Nicotiana tabacum), and of the PmH20 alpha- and beta-megaspermin, two elicitins of class I-A and I-B, respectively. The structure of the glycoprotein shows a signal peptide of 20 amino acids followed by the typical elicitin 98-amino acid-long domain and a 77-amino acid-long C-terminal domain carrying an O-glycosylated moiety. The molecular mass deduced from the translated cDNA sequence is 14,920 and 18,676 D as determined by mass spectrometry. This structure together with multiple sequence alignments and phylogenetic analyses indicate that the glycoprotein belongs to class III elicitins. It is the first class III elicitin protein characterized, which we named gamma-megaspermin. We compared the biological activity of the three PmH20 elicitins when applied to tobacco cv Samsun NN plants. Although alpha- and gamma-megaspermin were similarly active, beta-megaspermin was the most active in inducing the hypersensitive response and localized acquired resistance, which was assessed by measuring the levels of acidic and basic pathogenesis-related proteins and of the antioxidant phytoalexin scopoletin. The three elicitins induced similar levels of systemic acquired resistance measured as the expression of acidic PR proteins and is increased resistance to challenge tobacco mosaic virus infection.

Detection of resistance to fungicides, mating types and fitness of Phytophthora infestans in Hebei, China.

In vitro, isolates resistant to metalaxyl (M) and oxadixyl (O) of Phytophthora infestans were 11.2% of 62 isolates from potato and tomato in Hebei Province, mean resistance factor was 15,022 fold and 24,733 fold, respectively, no isolates resistant either to cymoxanil (C) or to dimethomorph (D) were detected. On the other hand, in vivo, isolates resistant to M and O were 29.0% and 32.7%, respectively, among 217 isolates from potato plants in Weichang and Chongli, Hebei, only one isolate resistant to M and O was found among 88 isolates from tomato plants in Xushui, Hebei. Among 73 isolates from potato in Weichang and Chongli, 6 isolates with A2 mating type were 8.2%, 3 A2 isolates resistant to metalaxyl and oxadixyl. 3 A2 isolates were 6.8% of 44 isolates from tomato in Xushui. Cross resistance in Phytophthora infestans was confirmed between M and O, but no cross resistance between C or D and M or O. The field isolates resistant to M and O were outstandingly fitter than sensitive or intermediate ones, mainly due to stronger sporulation capacity of the field isolates.