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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.

Phytophthora sojae is an oomycete and a pathogen of soybean that causes root rot. During infection P. sojae delivers effector proteins into host cells to foster disease. However, effector-triggered immunity (ETI) results when pathogen factors are recognized by host resistance (R) proteins. We have now identified the P. sojae Avr1d gene, which encodes a predicted effector protein with the amino acid motif Arg-X-Leu-Arg (RXLR). Genetic mapping of 16 different P. sojae isolates and of a segregating F2 population of 40 individuals shows that the predicted RXLR effector gene Avh6 precisely cosegregates with the Avr1d phenotype. Transient expression assays confirm that Avr1d triggers cell death specifically in Rps1d soybean plants. The Avr1d gene is present in P. sojae strains that are avirulent on Rps1d, whereas the gene is deleted from the genome of virulent strains. Two sequence variants of the Avr1d gene encoding different protein products occur in P. sojae strains, but both are recognized by Rps1d and cause ETI. Liposome binding assays show that Avr1d has affinity for phosphatidylinositol 4-phosphate and that binding can be disrupted by mutation of lysine residues in the carboxy-terminal effector domain of the protein. The identification of Avr1d aids pathogen diagnostics and soybean cultivar development.

Necrosis- and ethylene-inducing-like proteins (NLP) are widely distributed in eukaryotic and prokaryotic plant pathogens and are considered to be important virulence factors. We identified, in total, 70 potential Phytophthora sojae NLP genes but 37 were designated as pseudogenes. Sequence alignment of the remaining 33 NLP delineated six groups. Three of these groups include proteins with an intact heptapeptide (Gly-His-Arg-His-Asp-Trp-Glu) motif, which is important for necrosis-inducing activity, whereas the motif is not conserved in the other groups. In total, 19 representative NLP genes were assessed for necrosis-inducing activity by heterologous expression in Nicotiana benthamiana. Surprisingly, only eight genes triggered cell death. The expression of the NLP genes in P. sojae was examined, distinguishing 20 expressed and 13 nonexpressed NLP genes. Real-time reverse-transcriptase polymerase chain reaction results indicate that most NLP are highly expressed during cyst germination and infection stages. Amino acid substitution ratios (Ka/Ks) of 33 NLP sequences from four different P. sojae strains resulted in identification of positive selection sites in a distinct NLP group. Overall, our study indicates that expansion and pseudogenization of the P. sojae NLP family results from an ongoing birth-and-death process, and that varying patterns of expression, necrosis-inducing activity, and positive selection suggest that NLP have diversified in function.

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.

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.

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.

Six unique expressed sequence tag (EST) libraries were generated from four developmental stages of Phytophthora sojae P6497. RNA was extracted from mycelia, swimming zoospores, germinating cysts, and soybean (Glycine max (L.) Merr.) cv. Harosoy tissues heavily infected with P. sojae. Three libraries were created from mycelia growing on defined medium, complex medium, and nutrient-limited medium. The 26,943 high-quality sequences obtained clustered into 7,863 unigenes composed of 2,845 contigs and 5,018 singletons. The total number of P. sojae unigenes matching sequences in the genome assembly was 7,412 (94%). Of these unigenes, 7,088 (90%) matched gene models predicted from the P. sojae sequence assembly, but only 2,047 (26%) matched P. ramorum gene models. Analysis of EST frequency from different growth conditions and morphological stages revealed genes that were specific to or highly represented in particular growth conditions and life stages. Additionally, our results indicate that, during infection, the pathogen derives most of its carbon and energy via glycolysis of sugars in the plant. Sequences identified with putative roles in pathogenesis included avirulence homologs possessing the RxLR motif, elicitins, and hydrolytic enzymes. This large collection of P. sojae ESTs will serve as a valuable public genomic resource.

The year 2004 was an exciting one for the Phytophthora research community. The United States Department of Energy Joint Genome Institute (JGI) completed the draft genome sequence of two Phytophthora species, Phytophthora sojae and Phytophthora ramorum. In August of that year over 50 people gathered at JGI in Walnut Creek, California, for an annotation jamboree and searched for the secrets and surprises that the two genomes have in petto. This culminated in a paper in Science in September of this year describing the highlights of the sequencing project and emphasizing the power of having the genome sequences of two closely related organisms. This MPMI Focus issue on Phytophthora genomics contains a number of more specialized manuscripts centered on gene annotation and genome organization, and complemented with manuscripts that rely on genomics resources.

To investigate patterns of gene expression in soybean (Glycine max) and Phytophthora sojae during an infection time course, we constructed a 4,896-gene microarray of host and pathogen cDNA transcripts. Analysis of rRNA from soybean and P. sojae was used to estimate the ratio of host and pathogen RNA present in mixed samples. Large changes in this ratio occurred between 12 and 24 h after infection, reflecting the rapid growth and proliferation of the pathogen within host tissues. From the microarray analysis, soybean genes that were identified as strongly upregulated during infection included those encoding enzymes of phytoalexin biosynthesis and defense and pathogenesis-related proteins. Expression of these genes generally peaked at 24 h after infection. Selected lipoxygenases and peroxidases were among the most strongly downregulated soybean genes during the course of infection. The number of pathogen genes expressed during infection reached a maximum at 24 h. The results show that it is possible to use a single microarray to simultaneously probe gene expression in two interacting organisms. The patterns of gene expression we observed in soybean and P. sojae support the hypothesis that the pathogen transits from biotrophy to necrotrophy between 12 and 24 h after infection.

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.

The resistance of soybean to Phytophthora root and stem rot caused by Phytophthora sojae is conferred by a series of single-dominant Rps genes. We have applied random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) analyses to isolate molecular markers linked to Rps1-k. Five RAPD markers were identified and mapped closely to one side of Rps1-k. AFLP analysis was carried out with near isogenic lines and bulks obtained from F3 families. Twenty-seven markers were identified. Nineteen of these were specific to the resistant parent. Five AFLP markers were amplified from the susceptible parent. One of these markers, TC1, mapped at 0.07 centimorgans (cM) from the Rps1 locus. Three AFLP markers were co-dominant, and one of these, CG1, mapped at a distance of 0.06 cM from the Rps1 locus on the opposite side of the rest of the markers. Two RAPD, 17 AFLP, and 14 restriction fragment length polymorphism (RFLP) markers originating from duplicated sequences were mapped within a 3-cM map interval. These results suggest that Rps1-k is located at the end of an introgressed region. Physical mapping data indicate that the Rps1-k-flanking markers CG1 and TC1 may be located within a 125-kb chromosomal fragment.

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.

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 F(2) 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 co-bombardment with a reporter gene confirms that Avr3a/5 triggers cell death in Rps5 soybean leaves in an appropriate allele-specific manner. Sequence analysis of the Avr3a/5 gene identifies crucial residues in the effector domain that distinguish recognition by Rps3a and Rps5.

The importance of segmental duplications and copy number variants as a source of genetic and phenotypic variation is gaining greater appreciation, in a variety of organisms. Now, we have identified the Phytophthora sojae avirulence genes Avr1a and Avr3a and demonstrate how each of these Avr genes display copy number variation in different strains of P. sojae. The Avr1a locus is a tandem array of four near-identical copies of a 5.2 kb DNA segment. Two copies encoding Avr1a are deleted in some P. sojae strains, causing changes in virulence. In other P. sojae strains, differences in transcription of Avr1a result in gain of virulence. For Avr3a, there are four copies or one copy of this gene, depending on the P. sojae strain. In P. sojae strains with multiple copies of Avr3a, this gene occurs within a 10.8 kb segmental duplication that includes four other genes. Transcriptional differences of the Avr3a gene among P. sojae strains cause changes in virulence. To determine the extent of duplication within the superfamily of secreted proteins that includes Avr1a and Avr3a, predicted RXLR effector genes from the P. sojae and the P. ramorum genomes were compared by counting trace file matches from whole genome shotgun sequences. The results indicate that multiple, near-identical copies of RXLR effector genes are prevalent in oomycete genomes. We propose that multiple copies of particular RXLR effectors may contribute to pathogen fitness. However, recognition of these effectors by plant immune systems results in selection for pathogen strains with deleted or transcriptionally silenced gene copies.

Root and stem rot disease of soybean is caused by the oomycete Phytophthora sojae. The avirulence (Avr) genes of P. sojae control race-cultivar compatibility. In this study, we identify the P. sojae Avr3c gene and show that it encodes a predicted RXLR effector protein of 220 amino acids. Sequence and transcriptional data were compared for predicted RXLR effectors occurring in the vicinity of Avr4/6, as genetic linkage of Avr3c and Avr4/6 was previously suggested. Mapping of DNA markers in a F(2) population was performed to determine whether selected RXLR effector genes co-segregate with the Avr3c phenotype. The results pointed to one RXLR candidate gene as likely to encode Avr3c. This was verified by testing selected genes by a co-bombardment assay on soybean plants with Rps3c, thus demonstrating functionality and confirming the identity of Avr3c. The Avr3c gene together with eight other predicted genes are part of a repetitive segment of 33.7 kb. Three near-identical copies of this segment occur in a tandem array. In P. sojae strain P6497, two identical copies of Avr3c occur within the repeated segments whereas the third copy of this RXLR effector has diverged in sequence. The Avr3c gene is expressed during the early stages of infection in all P. sojae strains examined. Virulent alleles of Avr3c that differ in amino acid sequence were identified in other strains of P. sojae. Gain of virulence was acquired through mutation and subsequent sequence exchanges between the two copies of Avr3c. The results illustrate the importance of segmental duplications and RXLR effector evolution in the control of race-cultivar compatibility in the P. sojae and soybean interaction.

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Abstract Isoflavonoids are biologically active natural products that accumulate in soybean seeds during development. The amount of isoflavonoids present in soybean seed is variable, depending on genetic and environmental factors that are not fully understood. Experiments were conducted to determine whether isoflavonoids are synthesized within seed tissues during development, or made in other plant organs and transported to the seeds where they accumulate. An analysis of isoflavonoids by HPLC detected the compounds in all organs of soybean plant, but the amount of isoflavonoids present varied depending on the tissue and developmental stage. The greatest concentrations were found in mature seeds and leaves. The 2-hydroxyisoflavanone synthase genes IFS1 and IFS2 were studied to determine their pattern of expression in different tissues and developmental stages. The highest level of expression of IFS1 was observed in the root and seed coat, while IFS2 was most highly expressed in embryos and pods, and in elicitor-treated or pathogen-challenged tissues. Incorporation of radiolabel into isoflavonoids was observed when developing embryos and other plant organs were fed with [14C]phenylalanine. Embryos excised from developing soybean seeds also accumulated isoflavonoids from a synthetic medium. A maternal effect on seed isoflavonoid content was noted in reciprocal crosses between soybean cultivars that differ in seed isoflavonoids. From these results, we propose that developing soybean embryos have an ability to synthesize isoflavonoids de novo, but that transport from maternal tissues may in part contribute to the accumulation of these natural products in the seed. [ABSTRACT FROM AUTHOR]

Although the development of the soybean ovule has been fairly well studied, knowledge of the sequence of events in the seed coat during the first 3 weeks after flowering is incomplete. The goal of the present study was to document, using light microscopy, the early development of the soybean seed coat with respect to changes in structure and histochemistry. At anthesis, the seed coat consists of an outer layer of cuboidal epidermal cells surrounding several layers of undifferentiated parenchyma (which together constitute the outer integument), and an inner layer of cuboidal endothelial cells (the inner integument). At 3 d post anthesis (dpa), the inner integument has expanded to include three to five layers of relatively large cells with thick, heavily-staining cell walls immediately adjacent to the endothelium. By 18 dpa, the outer integument has developed into a complex of tissues comprised of an inner layer of thick-walled parenchyma, an outer layer of thin-walled parenchyma containing vascular tissue which has grown down from the lateral vascular bundles in the hilum region, a hypodermis of hourglass cells, and palisade layer (epidermis). The thick-walled parenchyma of the inner integument has become completely stretched and compressed, leaving a single, deeply staining wall layer directly above the endothelium. At 21 dpa, the outermost cells of the endosperm have begun to compress the endothelium. At 45 dpa (physiological maturity) the seed coat retains only the palisade layer, hourglass cells, and a few layers of thin-walled parenchyma. The innermost layer of the endosperm, the aleurone layer, adheres to the inside of the seed coat. This knowledge will be invaluable in future studies of manipulation of gene expression in the seed coat to modify seed or seed coat characteristics. Copyright 1999 Annals of Botany Company [ABSTRACT FROM AUTHOR]