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The use of resistance genes in elite soybean cultivars is one of the most widely used methods to manage Phytophthora sojae. This method relies on effector-triggered immunity, where a Resistant to P. sojae (Rps) gene product from the plant recognizes a specific effector from the pathogen, encoded by an avirulence (Avr) gene. Many Avr genes from P. sojae have been identified in the last decade, allowing a better exploitation of this type of resistance. The objective of the present study was to identify the Avr gene triggering immunity derived from the soybean resistance gene Rps8. The analysis of a segregating F

Oomycetes form a deep lineage of eukaryotic organisms that includes a large number of plant pathogens which threaten natural and managed ecosystems. We undertook a survey to query the community for their ranking of plant-pathogenic oomycete species based on scientific and economic importance. In total, we received 263 votes from 62 scientists in 15 countries for a total of 33 species. The Top 10 species and their ranking are: (1) Phytophthora infestans; (2, tied) Hyaloperonospora arabidopsidis; (2, tied) Phytophthora ramorum; (4) Phytophthora sojae; (5) Phytophthora capsici; (6) Plasmopara viticola; (7) Phytophthora cinnamomi; (8, tied) Phytophthora parasitica; (8, tied) Pythium ultimum; and (10) Albugo candida. This article provides an introduction to these 10 taxa and a snapshot of current research. We hope that the list will serve as a benchmark for future trends in oomycete research.

Cyclophilins (CYPs) belong to the immunophilin superfamily with peptidyl-prolyl cis-trans isomerase (PPIase) activity. They catalyze the interconversion of the cis- and trans-rotamers of the peptidyl-prolyl amide bond of peptides. A yeast-two-hybrid screening using the isoflavonoid regulator GmMYB176 as bait identified GmCYP1 as one of the interacting proteins in soybean embryos. GmCYP1 localizes both in the nucleus and cytoplasm, and interacts in planta with GmMYB176, in the nucleus, and with SGF14l (a soybean 14-3-3 protein) in the nucleus and the cytoplasm. GmCYP1 contains a single cyclophilin-like domain and displays a high sequence identity with other plant CYPs that are known to have stress-specific function. Tissue-specific expression of GmCYP1 revealed higher expression in developing seeds compared to other vegetative tissues, suggesting their seed-specific role. Furthermore, GmCYP1 transcript level was reduced in response to stress. Since isoflavonoids are involved in plant stress resistance against biotic and abiotic factors, the interaction of GmCYP1 with the isoflavonoid regulators GmMYB176 and 14-3-3 protein suggests its role in defense in soybean.

The process of RNA splicing influences many physiological processes, including plant immunity. However, how plant parasites manipulate host RNA splicing process remains unknown. Here we demonstrate that PsAvr3c, an avirulence effector from oomycete plant pathogen Phytophthora sojae, physically binds to and stabilizes soybean serine/lysine/arginine-rich proteins GmSKRPs. The SKRPs are novel proteins that associate with a complex that contains plant spliceosome components, and are negative regulators of plant immunity. Analysis by RNA-seq data indicates that alternative splicing of pre-mRNAs from 401 soybean genes, including defense-related genes, is altered in GmSKRP1 and PsAvr3c overexpressing lines compared to control plants. Representative splicing events mediated by GmSKRP1 and PsAvr3c are tested by infection assays or by transient expression in soybean plants. Our results show that plant pathogen effectors can reprogram host pre-mRNA splicing to promote disease, and we propose that pathogens evolved such strategies to defeat host immune systems., Various effectors of plant pathogens modulate host cell biology. Here, Huang et al. show PsAvr3c, an avirulence effector from oomycete plant pathogen Phytophthora sojae, can reprogram host pre-mRNA splicing for immune modulation.

Ranathunge, K., Thomas, R. H., Fang, X., Peterson, C. A., Gijzen, M., and Bernards, M. A. 2008. Soybean root suberin and partial resistance to root rot caused by Phytophthora sojae. Phytopathology 98:1179-1189. Phytophthora sojae is the causal agent of root and stem rot of soybean (Glycine max). Various cultivars with partial resistance to the pathogen have been developed to mitigate this damage. Herein, two contrasting genotypes, the cultivar Conrad (with strong partial resistance) and the line OX760-6 (with weak partial resistance), were compared regarding their amounts of preformed and induced suberin components, and to early events during the P. sojae infection process. To colonize the root, hyphae grew through the suberized middle lamellae between epidermal cells. This took 2 to 3 h longer in Conrad than in OX760-6, giving Conrad plants more time to establish their chemical defenses. Subsequent growth of hyphae through the endodermis was also delayed in Conrad. This cultivar had more preformed aliphatic suberin than the line OX760-6 and was induced to form more aliphatic suberin several days prior to that of OX760-6. However, the induced suberin was formed subsequent to the initial infection process. Eventually, the amount of induced suberin (measured 8 days postinoculation) was the same in both genotypes. Preformed root epidermal suberin provides a target for selection and development of new soybean cultivars with higher levels of expression of partial resistance to P. sojae.

Soybean root fluorescence in ultraviolet light is controlled by the Fr loci. Extracts from fluorescent roots of soybean contain three closely related fluorescent compounds that are greatly reduced in extracts from non-fluorescent roots. Root fluorescence is due to their presence, regardless of the Frlocus that conditions this trait. Key words: Coumarin, genetic locus, glycoside, fluorescence, roots, soybean

The Phytophthora sojae avirulence gene Avr3a encodes an effector that is capable of triggering immunity on soybean plants carrying the resistance gene Rps3a. P. sojae strains that express Avr3a are avirulent to Rps3a plants, while strains that do not are virulent. To study the inheritance of Avr3a expression and virulence towards Rps3a, genetic crosses and self-fertilizations were performed. A cross between P. sojae strains ACR10 X P7076 causes transgenerational gene silencing of Avr3a allele, and this effect is meiotically stable up to the F5 generation. However, test-crosses of F1 progeny (ACR10 X P7076) with strain P6497 result in the release of silencing of Avr3a. Expression of Avr3a in the progeny is variable and correlates with the phenotypic penetrance of the avirulence trait. The F1 progeny from a direct cross of P6497 X ACR10 segregate for inheritance for Avr3a expression, a result that could not be explained by parental imprinting or heterozygosity. Analysis of small RNA arising from the Avr3a gene sequence in the parental strains and hybrid progeny suggests that the presence of small RNA is necessary but not sufficient for gene silencing. Overall, we conclude that inheritance of the Avr3a gene silenced phenotype relies on factors that are variable among P. sojae strains.

Hydrophobic protein from soybean (HPS) is present in soybean dust and is an allergen (Gly m 1) that causes asthma in allergic individuals. Past studies have shown that HPS occurs on the seed surface. To determine the microscopic localization of HPS during seed development, monoclonal antibodies to HPS were used to visualize the protein by fluorescence and transmission electron microscopy. Seed coat and endocarp sections were also examined for pectin, cellulose, callose, starch and protein by histochemical staining. HPS is present in the endocarp epidermal cells at 18 to 28 days post anthesis. At later stages of seed development, HPS occurs in extracellular secretions that accumulate unevenly on the endocarp epidermis and seed surface. HPS is synthesized by the endocarp epidermis and deposited on the seed surface as part of a heterogeneous matrix.

We have used cDNA microarray analysis to examine changes in gene expression during embryo development in soybean (Glycine max) and to compare gene expression profiles of two soybean cultivars that differ in seed isoflavonoid content. The analysis identified 5,910 genes that were differentially expressed in both soybean cultivars grown at two different locations for two consecutive years in one of the five different stages of embryo development. An ANOVA analysis with P value < 0.05 and < 0.01 indicated that gene expression changes due to environmental factors are greater than those due to cultivar differences. Most changes in gene expression occurred at the stages when the embryos were at 30 or 70 d after pollination. A significantly larger fraction of genes (48.5%) was expressed throughout the development and showed little or no change in expression. Transcript accumulation for genes related to the biosynthesis of storage components in soybean embryos showed several unique temporal expressions. Expression patterns of several genes involved in isoflavonoid biosynthesis, such as Phenylalanine Ammonia-Lyase, Chalcone Synthase (CHS) 7, CHS8, and Isoflavone Synthase2, were higher at 70 d after pollination in both the cultivars. Thus, expression of these genes coincides with the onset of accumulation of isoflavonoids in the embryos. A comparative analysis of genes involved in isoflavonoid biosynthesis in RCAT Angora (high seed isoflavonoid cultivar) and Harovinton (low seed isoflavonoid cultivar) revealed that CHS7 and CHS8 were expressed at significantly greater level in RCAT Angora than in Harovinton. Our study provides a detailed transcriptome profiling of soybean embryos during development and indicates that differences in the level of seed isoflavonoids between these two cultivars could be as a result of differential expression of CHS7 and CHS8 during late stages of seed development.

The seed coat serves as a multifunctional organ with a role in protection and for the supply of nutrients to the embryo sac during development. The composition of the legume seed coat differs from other seed tissues in many ways including its protein composition. An abundant 24 kDa protein (SC24) has been purified and identified from soybean (Glycine max [L.] Merr) seed hulls. The corresponding cDNA and genomic DNA clones for SC24 were isolated and characterized, and expression patterns were determined. The deduced protein sequence of 219 amino acids included an N-terminal signal peptide. Transcripts encoding SC24 were present in the seed coat from 30 days after pollination (DAP) until maturity, but the protein was not detected until the final stages of seed maturation. In mature seeds, most of the SC24 protein was localized to the parenchyma and aleurone layers of the seed coat. The expression of SC24 was also induced in vegetative tissues by pathogen infection and by wounding. The SC24 protein bound to an affinity column containing an isophthalic acid ligand, and was eluted with 7 mM citrate. Polyclonal antibodies raised against recombinant SC24 cross-reacted with the seed coat peroxidase enzyme, suggesting that these two proteins may share an antigenic determinant. Overall, the results indicate that SC24 belongs to a novel class of plant defence proteins with carboxylate-binding activity.

Soybean peroxidase (SBP), (EC 1.11.1.7) can be readily extracted from soybean seed hulls. This study reports on the direct use of soybean seed-hull extracts for the bioremediation of phenolic wastes. The crude SBP extract from the hulls, like pure soybean peroxidase, is catalytically active in a broad range of pH and temperatures. As SBP is gradually released into the aqueous solution from seed hulls, the direct use of soybean seed hulls can reduce SBP inactivation by H 2 O 2 and enhance the utilization efficiency of SBP through the slow release of the enzyme from the seed hulls. However, large doses of soybean seed hulls were found to be ineffective in phenol removal. Gradual additions of H 2 O 2 in combination with the SBP released from the hulls were applied to optimize the bioremediation. Since the crude extract contains a mixture of multiple soybean proteins, soybean seed hull slurry required a higher concentration of H 2 O 2 to remove the phenolic substrates than did the purified enzyme. Under the experimental conditions, 80 % of phenol (10.6 mM), 96 % of 2-chlorophenol (3.9 mM), 95 % of 2,4-dichlorophenol (3.1 mM), and 94 % of mixed phenol and chlorophenols were removed using soybean seed hulls in a single batch reactor. These results demonstrate that soybean seed hulls, compared to purified SBP, may be a more cost-effective alternative in the enzymatic removal of phenolic compounds through polymerization reactions.

SUMMARY Transcripts encoding elicitin-like protein domains were identified from similarity searches of Phytophthora sojae expressed sequence tags and were characterized with regard to molecular structure and elicitor activity. The P. sojae elicitin family consists of at least nine genes with products similar to previously described elicitins (SOJA-2, SOJB, SOJ2, SOJ3, SOJ5, SOJ6 and SOJ7) or highly diverged from known sequences (SOJX and SOJY). The predicted structural features of seven (SOJA-2, SOJB, SOJ2, SOJ3, SOJ6, SOJX and SOJY) of the elicitin preproteins were compared. All of the predicted elicitins possess a leader signal sequence and a core elicitin domain. Five (SOJ2, SOJ3, SOJ6, SOJX and SOJY) of the characterized elicitins also contain a variable C-terminal region. In addition, SOJX and SOJY contain a C-terminal hydrophobic membrane-spanning domain. An analysis of expression patterns of the elicitin transcripts showed that SOJA-2, SOJB, SOJ2, SOJ3 and SOJ6 were expressed in axenically grown mycelia and during infection, but not in zoospores. In contrast, SOJX and SOJY were predominantly and specifically expressed in zoospores. Selected elicitin domains were also tested for the induction of the hypersensitive response (HR) in Nicotiana spp. All of the elicitin protein domains tested induced the HR, except for SOJX and SOJY. Overall, the results show that the P. sojae elicitin gene family is large and diverse, with varying patterns of expression and HR-inducing activity.

The 90-kD molecular chaperone hsp90 is the key component of a multiprotein chaperone complex that facilitates folding, stabilization, and functional modulation of a number of signaling proteins. The components of the animal chaperone complex include hsp90, hsp70, hsp40, Hop, and p23. The animal Hop functions to link hsp90 and hsp70, and it can also inhibit the ATPase activity of hsp90. We have demonstrated the presence of an hsp90 chaperone complex in plant cells, but not all components of the complex have been identified. Here, we report the isolation and characterization of soybean (Glycine max) GmHop-1, a soybean homolog of mammalian Hop. An analysis of soybean expressed sequence tags, combined with preexisting data in literature, suggested the presence of at least three related genes encoding Hop-like proteins in soybean. Transcripts corresponding to Hop-like proteins in soybean were detected under normal growth conditions, and their levels increased further in response to stress. A recombinant GmHop-1 bound hsp90 and its binding to hsp90 could be blocked by the tetratricopeptide repeat (TPR) domain of rat (Rattus norvegicus) protein phosphatase 5. Deletion of amino acids 325 to 395, adjacent to the TPR2A domain in GmHop-1, resulted in loss of hsp90 binding. In a minimal assembly system, GmHop-1 was able to stimulate mammalian steroid receptor folding. These data show that plant and animal Hop homologs are conserved in their general characteristics, and suggest that a Hop-like protein in plants is an important cochaperone of plant hsp90.

Plant pathogens display impressive versatility in adapting to host immune systems. Pathogen effector proteins facilitate disease but can become avirulence (Avr) factors when the host acquires discrete recognition capabilities that trigger immunity. The mechanisms that lead to changes to pathogen Avr factors that enable escape from host immunity are diverse, and include epigenetic switches that allow for reuse or recycling of effectors. This perspective outlines possibilities of how epigenetic control of Avr effector gene expression may have arisen and persisted in filamentous plant pathogens, and how it presents special problems for diagnosis and detection of specific pathogen strains or pathotypes.

Soybean seed hull peroxidase (SBP) is an inexpensive oxidoreductive enzyme and could potentially be used to oxidise/polymerise various organic pollutants present in the industrial and petrochemical wastes. SBP is able to retain its catalytic properties under wide ranges of pH and at elevated temperatures. In this study, a systematic evaluation of the biocatalytic properties of SBP was carried out. The optimal pH for SBP activity is pH 6.0 and significant activity was observed between 2.2 and 8.0. SBP also showed three times higher activity at an elevated temperature of 80°C and at pH 6.0 when compared to the activity at room temperature. The pH and temperature of the reaction mixture were found to significantly influence the SBP activity. SBP is fairly active in organic solvents. The enzyme exhibited highest activity in the presence of 16.67% (w/v) ethanol followed by acetone, methanol and acetonitrile. The enzyme activity was reduced with an increase in concentration of the organic solvent. SBP also showed maximum activity at different concentrations of acetone using a phosphate buffer, pH 6.0 than with the other pH buffers. Benzene/acetone mixture seems to be another better solvent system for SBP where it showed about 65% of its activity at 16.67% (w/v) concentration.

Phytophthora sojae (Kaufmann and Gerdemann) is an oomycete that causes stem and root rot on soybean (Glycine max L. Merr) plants. We have constructed three cDNA libraries using mRNA isolated from axenically grown mycelium and zoospores and from tissue isolated from plant hypocotyls 48 h after inoculation with zoospores. A total of 3,035 expressed sequence tags (ESTs) were generated from the three cDNA libraries, representing an estimated 2,189 cDNA transcripts. The ESTs were classified according to putative function based on similarity to known proteins, and were analyzed for redundancy within and among the three source libraries. Distinct expression patterns were observed for each library. By analysis of the percentage G+C content of the ESTs, we estimate that two-thirds of the ESTs from the infected plant library are derived from P. sojae cDNA transcripts. The ESTs originating from this study were also compared with a collection of Phytophthora infestans ESTs and with all other non-human ESTs to assess the similarity of the P. sojae sequences to existing EST data. This collection of cDNA libraries, ESTs, and accompanying annotation will provide a new resource for studies on oomycetes and on soybean responses to pathogen challenge.

A seed coat-specific gene, SCS1 (Seed Coat Subtilisin 1), from soybean, Glycine max [L.] Merill, has been identified and studied. The gene belongs to a small family of genes with sequence similarity to the subtilisins, which are serine proteases. Northern blot analysis showed that SCS1 RNA accumulates to maximal levels in seed coats at 12 days post anthesis, preceding the final stages of seed coat differentiation. The SCS1 RNA was not found in other tissues including embryos, seed pods, flowers, stems, roots or leaves. In-situ hybridization studies confirmed the temporal pattern of expression observed by Northern blot analysis and further revealed a restricted pattern of RNA accumulation in thick-walled parenchyma cells of the seed coats. These cells are important in the apoplastic translocation of nutrients en route to the embryo from the vascular tissues. The tissue-specific subtilisin-like gene may be required for regulating the differentiation of the thick-walled parenchyma cells.

Stevia rebaudiana Bertoni leaves accumulate a mixture of at least eight different glycosides derived from the tetracyclic diterpene steviol. These natural products taste intensely sweet and have similar biosynthetic origins to those of gibberellic acid (GA). The initial steps leading to the formation of GA result from the two-step cyclization of geranylgeranyl diphosphate (GGDP) to (-)-kaurene via the action of two terpene cyclases (-)-copalyl diphosphate synthase (CPS) and (-)-kaurene synthase (KS). Steviol biosynthesis probably uses the same mechanism although the genes and enzymes from S. rebaudiana that are involved in the cyclization of GGDP have not been characterized. We have isolated both the CPS and KS genes from S. rebaudiana and found that recombinant CPS and KS were catalytically active, suggesting that the CPS and KS genes participate in steviol biosynthesis. The genes coding for CPS and KS are usually present in single copies in most plant species and their expression is normally low and limited to rapidly growing tissues. The KS gene has been duplicated in the S. rebaudiana genome and both the KS and CPS genes are highly expressed in mature leaves, a pattern opposite to that found with GA biosynthesis. This pattern may, at least in part, lead to temporal and spatial separation of GA and steviol biosynthesis and probably helps to prevent over-expression from interfering with normal GA metabolism. Our results show that CPS and KS are part of the steviol glycoside biosynthetic pathway and that Stevia rebaudiana has recruited two genes to secondary metabolism from a highly regulated pathway involved in hormone biosynthesis.

Stevia rebaudiana is a member of the Compositae, native to Paraguay. It produces a number of high-potency low-calorie sweeteners in its leaf tissue. The sweeteners are diterpene glycosides and range between 30 and 320 times sweeter than sugar. Increasing consumer interest in natural food ingredients means that products like stevia sweeteners will be subject to increasing demand. Such demand will need to be supported by a modern mechanised production system. The purpose of this review is to summarize the existing agricultural, chemical and biochemical literature to provide a baseline for new research. Key words: Stevia, diterpene, steviol glycoside, sweeteners

Grapevine (Vitis vinifera) is susceptible to many pathogens which cause significant losses to viticulture worldwide. Chemical control is available, but agro-ecological concerns have raised interest in alternative methods, especially in triggering plant immunity by elicitor treatments. The b-glucan laminarin (Lam) and its sulfated derivative (PS3) have been previously demonstrated to induce resistance in grapevine against downy mildew (Plasmopara viticola). However, if Lam elicits classical grapevine defenses such as oxidative burst, pathogenesis-related (PR)-proteins and phytoalexin production, PS3 triggered grapevine resistance via a poorly understood priming phenomenon. The aim of this study was to identify the molecular mechanisms of the PS3-induced resistance. For this purpose we studied i) the signaling events and transcriptome reprogramming triggered by PS3 treatment on uninfected grapevine, ii) grapevine immune responses primed by PS3 during P. viticola infection. Our results showed that i) PS3 was unable to elicit reactive oxygen species (ROS) production, cytosolic Ca2+ concentration variations, mitogen-activated protein kinase (MAPK) activation but triggered a long lasting plasma membrane depolarization in grapevine cells, ii) PS3 and Lam shared a common stress-responsive transcriptome profile that partly overlapped the salicylate- (SA) and jasmonate-(JA)-dependent ones. After P. viticola inoculation, PS3 specifically primed the SA- and ROS-dependent defense pathways leading to grapevine induced resistance against this biotroph. Interestingly pharmacological approaches suggested that the plasma membrane depolarization and the downstream ROS production are key events of the PS3-induced resistance

The aim of this work was to map and identify the Phytophthora sojae Avr1c gene. Progeny from a cross of P. sojae strains ACR10×P7076 were tested for virulence on plants carrying Rps1c. Results indicate that avirulence segregates as a dominant trait. We mapped the Avr1c locus by performing whole genome re-sequencing of composite libraries created from pooled samples. Sequence reads from avirulent (Pool1) and virulent (Pool2) samples were aligned to the reference genome and single nucleotide polymorphisms (SNP) were identified for each pool. High quality SNPs were filtered to select for positions where SNP frequency was close to expected values for each pool. Only three SNP positions fit all requirements, and these occurred in close proximity. Additional DNA markers were developed and scored in the F₂ progeny, producing a fine genetic map that places Avr1c within the Avr1a gene cluster. Transient expression of Avr1c or Avr1a triggers cell death on Rps1c plants, but Avr1c does not trigger cell death on Rps1a plants. Sequence comparisons show that the RXLR effector genes Avr1c and Avr1a are closely related paralogs. Gain of virulence on Rps1c in P. sojae strain P7076 is achieved by gene deletion, but in most other strains this is accomplished by gene silencing. This work provides practical tools for crop breeding and diagnostics, as the Rps1c gene is widely deployed in commercial soybean cultivars.

Ash dieback is a devastating fungal disease of ash trees that has swept across Europe and recently reached the UK. This emergent pathogen has received little study in the past and its effect threatens to overwhelm the ash population. In response to this we have produced some initial genomics datasets and taken the unusual step of releasing them to the scientific community for analysis without first performing our own. In this manner we hope to ‘crowdsource’ analyses and bring the expertise of the community to bear on this problem as quickly as possible. Our data has been released through our website at oadb.tsl.ac.uk and a public GitHub repository.

A factor present in the granules of Trichoplusia ni granulosis virus, when fed together with a nuclear polyhedrosis virus, can enhance viral infection and accelerate death in T. ni neonate larvae. Occlusion bodies of T. ni granulosis virus were dissolved in alkali and soluble proteins were separated by gel filtration chromatography. These fractions were assayed for enhancement of nuclear polyhedrosis infection in T. ni neonates. Denaturing gel electrophoresis of the fractions revealed that enhancin, a protein homologous to the synergistic factor of Pseudaletia unipuncta, eluted as a discrete peak. Viral enhancing activity eluted over a large volume covering this peak and a wide molecular weight range following this peak. Moreover, we show that the 98-kDa enhancin protein is released from a larger 150-kDa complex, by heating in the presence of detergent. Once released, the enhancin protein is subject to extensive proteolysis, yielding smaller protein fragments. These results suggest the possibility that fragments of the enhancin protein, or other components of the 150-kDa complex, may be active viral enhancing agents.

Plants have evolved pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI) to protect themselves from infection by diverse pathogens. Avirulence (Avr) effectors that trigger plant ETI as a result of recognition by plant resistance (R) gene products have been identified in many plant pathogenic oomycetes and fungi. However, the virulence functions of oomycete and fungal Avr effectors remain largely unknown. Here, we combined bioinformatics and genetics to identify Avr3b, a new Avr gene from Phytophthora sojae, an oomycete pathogen that causes soybean root rot. Avr3b encodes a secreted protein with the RXLR host-targeting motif and C-terminal W and Nudix hydrolase motifs. Some isolates of P. sojae evade perception by the soybean R gene Rps3b through sequence mutation in Avr3b and lowered transcript accumulation. Transient expression of Avr3b in Nicotiana benthamiana increased susceptibility to P. capsici and P. parasitica, with significantly reduced accumulation of reactive oxygen species (ROS) around invasion sites. Biochemical assays confirmed that Avr3b is an ADP-ribose/NADH pyrophosphorylase, as predicted from the Nudix motif. Deletion of the Nudix motif of Avr3b abolished enzyme activity. Mutation of key residues in Nudix motif significantly impaired Avr3b virulence function but not the avirulence activity. Some Nudix hydrolases act as negative regulators of plant immunity, and thus Avr3b might be delivered into host cells as a Nudix hydrolase to impair host immunity. Avr3b homologues are present in several sequenced Phytophthora genomes, suggesting that Phytophthora pathogens might share similar strategies to suppress plant immunity., Author Summary Phytophthora, a group of notorious oomycete pathogens, damages a very wide range of crop, vegetable, pasture and horticultural plants, generating great losses to agricultural production annually. Disease outcomes between plants and Phytophthora pathogens often depend on whether plants carry resistance (R) gene-encoded receptors than recognize the presence of pathogen avirulence (Avr) effectors. Previous studies identified a conserved host-targeting motif, RXLR (arginine, any, leucine, arginine), common to several Phytophthora Avr effectors. The genome sequencing of several Phytophthora species including P. infestans (potato late blight pathogen) and P. sojae (soybean root rot disease pathogen), resulted in the identification of a large reservoir of RXLR-carrying effector candidates. In this paper we identified an RXLR-carrying protein from P. sojae as Avr effector Avr3b based on genetic mapping, sequence polymorphisms, and transient expression. Avr3b carries a Nudix hydrolase motif at its C-terminus and enhances Phytophthora virulence. Biochemical assays revealed that Avr3b is a pyrophosphorylase with ADP-ribose and NADH as its preferred substrates. Furthermore, the enzymatic activity is required for Avr3b to promote virulence but is not required for recognition by Rps3b.

Transglutaminases (TGases) are ubiquitous enzymes that catalyze selective cross-linking between protein-bound glutamine and lysine residues; the resulting isopeptide bond confers high resistance to proteolysis. Phytophthora sojae, a pathogen of soybean, secretes a Ca(2+)-dependent TGase (GP42) that is activating defense responses in both host and non-host plants. A GP42 fragment of 13 amino acids, termed Pep-13, was shown to be absolutely indispensable for both TGase and elicitor activity. GP42 does not share significant primary sequence similarity with known TGases from mammals or bacteria. This suggests that GP42 has evolved novel structural and catalytic features to support enzymatic activity. We have solved the crystal structure of the catalytically inactive point mutant GP42 (C290S) at 2.95 Å resolution and identified residues involved in catalysis by mutational analysis. The protein comprises three domains that assemble into an elongated structure. Although GP42 has no structural homolog, its core region displays significant similarity to the catalytic core of the Mac-1 cysteine protease from Group A Streptococcus, a member of the papain-like superfamily of cysteine proteases. Proteins that are taxonomically related to GP42 are only present in plant pathogenic oomycetes belonging to the order of the Peronosporales (e.g. Phytophthora, Hyaloperonospora, and Pythium spp.) and in marine Vibrio bacteria. This suggests that a lateral gene transfer event may have occurred between bacteria and oomycetes. Our results offer a basis to design and use highly specific inhibitors of the GP42-like TGase family that may impair the growth of important oomycete and bacterial pathogens.

The transcriptome of the oomycete plant pathogen Phytophthora sojae was profiled at ten different developmental and infection stages based on a 3′-tag digital gene-expression protocol. More than 90 million clean sequence tags were generated and compared with the P. sojae genome and its 19,027 predicted genes. A total of 14,969 genes were detected, of which 10,044 were deemed reliable because they mapped to unambiguous tags. A comparison of the whole-library genes' expression patterns suggested four groups: i) mycelia and zoosporangia, ii) zoospores and cysts, iii) germinating cysts, and iv) five infection site libraries (IF1.5 to IF24h). The libraries from the different groups showed major transitional shifts in gene expression. From the ten libraries, 722 gene expression–pattern clusters were obtained and the top 16 clusters, containing more than half of the genes, comprised enriched genes with different functions including protein localization, triphosphate metabolism, signaling process, and noncoding RNA metabolism. An evaluation of the average expression level of 30 pathogenesis-related gene families revealed that most were infection induced but with diverse expression patterns and levels. A web-based server named the Phytophthora Transcriptional Database has been established.

The perception of Phytophthora sojae avirulence (Avr) gene products by corresponding soybean resistance (Rps) gene products causes effector triggered immunity. Past studies have shown that the Avr3a and Avr5 genes of P. sojae are genetically linked, and the Avr3a gene encoding a secreted RXLR effector protein was recently identified. We now provide evidence that Avr3a and Avr5 are allelic. Genetic mapping data from F2 progeny indicates that Avr3a and Avr5 co-segregate, and haplotype analysis of P. sojae strain collections reveal sequence and transcriptional polymorphisms that are consistent with a single genetic locus encoding Avr3a/5. Transformation of P. sojae and transient expression in soybean were performed to test how Avr3a/5 alleles interact with soybean Rps3a and Rps5. Over-expression of Avr3a/5 in a P. sojae strain that is normally virulent on Rps3a and Rps5 results in avirulence to Rps3a and Rps5; whereas silencing of Avr3a/5 causes gain of virulence in a P. sojae strain that is normally avirulent on Rps3a and Rps5 soybean lines. Transient expression and cobombardment with a reporter gene confirms that Avr3a/5 triggers cell death in Rps5 soybean leaves in an appropriate allelespecific manner. Sequence analysis of the Avr3a/5 gene identifies crucial residues in the effector domain that distinguish recognition by Rps3a and Rps5. This work was supported by grants to MG from the Agriculture and Agri-Food Canada (AAFC) Crop Genomics Initiative; to SD from the National Natural Science Foundation of China (30900932); and to BMT from the Agriculture and Food Research Initiative of the USDA National Institute for Food and Agriculture, grant numbers 2007-35600-18530 and 2007-35319-18100, and from the US National Science Foundation (NSF), grant numbers MCB-0731969 and IOS-0924861. SD and DY were supported at AAFC by the China Scholarship Council. SDK was supported by an NSF pre-doctoral fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Two Soybean mosaic virus (SMV) G2 isolates, L and L-RB, sharing high-sequence similarly but differing in ability to break Rsv4-mediated resistance in soybean, were investigated. Infectious clones corresponding to these two isolates and their chimeric clones resulting from swapping different regions of genomic cDNA between L and L-RB were constructed. Only L-RB or chimeras containing the middle fragment of L-RB cDNA showed virulence on Rsv4–genotype soybean. Sequence comparison analysis revealed that the middle genomic region of L and L-RB encodes four different amino acids. Point mutagenesis demonstrated that a single amino acid substitution (Q1033K) in the P3 protein determined virulence toward Rsv4 resistance. In addition, six new SMV Rsv4 resistance-breaking isolates, variants of the second passage on Williams 82 infected with the chimeras or mutants noninfectious on soybean carrying Rsv4, were obtained. Sequencing data indicated that these new isolates contain either the Q1033K mutation or a new substitution (G1054R) in P3. Site-directed mutagenesis confirmed the virulence role of the G1054R mutation on Rsv4-genotype soybean. Taken together, these data suggest that P3 of the SMV G2 strain is an avirulent determinant for Rsv4 and one single nucleotide mutation in P3 may be sufficient to compromise its elicitor function.

SUMMARY Transcripts encoding elicitin-like protein domains were identified from similarity searches of Phytophthora sojae expressed sequence tags and were characterized with regard to molecular structure and elicitor activity. The P. sojae elicitin family consists of at least nine genes with products similar to previously described elicitins (SOJA-2, SOJB, SOJ2, SOJ3, SOJ5, SOJ6 and SOJ7) or highly diverged from known sequences (SOJX and SOJY). The predicted structural features of seven (SOJA-2, SOJB, SOJ2, SOJ3, SOJ6, SOJX and SOJY) of the elicitin preproteins were compared. All of the predicted elicitins possess a leader signal sequence and a core elicitin domain. Five (SOJ2, SOJ3, SOJ6, SOJX and SOJY) of the characterized elicitins also contain a variable C-terminal region. In addition, SOJX and SOJY contain a C-terminal hydrophobic membrane-spanning domain. An analysis of expression patterns of the elicitin transcripts showed that SOJA-2, SOJB, SOJ2, SOJ3 and SOJ6 were expressed in axenically grown mycelia and during infection, but not in zoospores. In contrast, SOJX and SOJY were predominantly and specifically expressed in zoospores. Selected elicitin domains were also tested for the induction of the hypersensitive response (HR) in Nicotiana spp. All of the elicitin protein domains tested induced the HR, except for SOJX and SOJY. Overall, the results show that the P. sojae elicitin gene family is large and diverse, with varying patterns of expression and HR-inducing activity.

Whether a seed coat of a soybean (Glycine max L. Mer.) seed is permeable or non-permeable is governed by a number of quantitative trait loci further influenced by environmental factors. In soybean seeds, water loss is controlled by a thin, inconspicuous outer cuticle. When intact, the outer cuticle constitutes a barrier to water passage; however, the presence of minute cracks in the cuticle results in the ready passage of water. We explored the timing of cuticular development in soybean seeds by measuring the deposition of the cutin in relation to seed growth and cell viability. Cutin deposition occurred early in the development and ceased just prior to the final stage of rapid seed expansion. Cracks in the cuticle appeared after cutin synthesis ceased while the seed continued to grow. In permeable seeds (regardless of genotype) the resistance of the cuticle to water passage increased steadily during development until seed expansion was maximal and cracks appeared in the cuticle. Once cracks formed, they became the primary site of water passage and the cuticle lost its ability to control the process. In non-permeable seeds, no cracks appeared at this critical point and the cuticle continued to restrict water passage. Microarray analysis of gene expression during seed coat development revealed a complex transcriptome with many genes uniquely expressed in the seed coat. However, the expression patterns were remarkably similar between permeable and non-permeable types, in keeping with the complexity of the underlying genetics of seed coat permeability.

Sequencing of the potato late blight pathogen Phytophthora infestans provides insight into the structure and evolution of its genome., Sequencing of the genome of the potato late blight pathogen Phytophthora infestans provides insight into genome structure and evolution within this genus of plant pathogenic oomycetes.