Mark Gijzen

Phytotoxicity and Innate Immune Responses Induced by Nep1-Like Proteins

We show that oomycete-derived Nep1 (for necrosis and ethylene-inducing peptide1)–like proteins (NLPs) trigger a comprehensive immune response in Arabidopsis thaliana, comprising posttranslational activation of mitogen-activated protein kinase activity, deposition of callose, production of nitric oxide, reactive oxygen intermediates, ethylene, and the phytoalexin camalexin, as well as cell death. Transcript profiling experiments revealed that NLPs trigger extensive reprogramming of the Arabidopsis transcriptome closely resembling that evoked by bacteria-derived flagellin. NLP-induced cell death is an active, light-dependent process requiring HSP90 but not caspase activity, salicylic acid, jasmonic acid, ethylene, or functional SGT1a/SGT1b. Studies on animal, yeast, moss, and plant cells revealed that sensitivity to NLPs is not a general characteristic of phospholipid bilayer systems but appears to be restricted to dicot plants. NLP-induced cell death does not require an intact plant cell wall, and ectopic expression of NLP in dicot plants resulted in cell death only when the protein was delivered to the apoplast. Our findings strongly suggest that NLP-induced necrosis requires interaction with a target site that is unique to the extracytoplasmic side of dicot plant plasma membranes. We propose that NLPs play dual roles in plant pathogen interactions as toxin-like virulence factors and as triggers of plant innate immune responses.

Patterns of gene expression upon infection of soybean plants by Phytophthora sojae

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 Top 10 oomycete pathogens in molecular plant pathology

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.

Isoflavonoid biosynthesis and accumulation in developing soybean seeds

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.

Copy Number Variation and Transcriptional Polymorphisms of Phytophthora sojae RXLR Effector Genes Avr1a and Avr3a

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.

Phytophthora sojae Avirulence Effector Avr3b is a Secreted NADH and ADP-ribose Pyrophosphorylase that Modulates Plant Immunity

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.

Transcriptome Analysis Reveals a Critical Role of CHS7 and CHS8 Genes for Isoflavonoid Synthesis in Soybean Seeds

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.

Removal of aqueous phenol and 2-chlorophenol with purified soybean peroxidase and raw soybean hulls

In this study, the removal of phenol and 2-chlorophenol using soybean seed-hulls in the presence of hydrogen peroxide is demonstrated. The performance of a stirred membrane reactor containing soluble purified SBP was compared with a batch stirred reactor containing raw soybean seed-hulls. The purified enzyme reactor proved to be ineffective while excellent results were obtained with the crude seed-hulls for the removal of phenol and 2-chlorophenol. Four sequential batch reactors containing raw seed-hulls achieved greater than 96% removal of phenol with a retention time of 20min in each reactor. A single batch reactor containing raw seed-hulls was effective in removing greater than 98.5% of 2-chlorophenol (initially at 1000 ppm) in less than 15 min. The performance of these reactors is comparable to existing HRP-based technology. The stability of the soybean peroxidase (SBP) enzyme was also examined in the presence of detergents (SDS, Tween 20 and Triton X-100). Low concentrations of the detergents significantly increased the enzyme activity and higher concentrations of detergents (up to 20% w/v) did not inactivate the SBP enzyme. These results demonstrate that SBP has good potential for the treatment of phenol contaminated solutions.

The Phytophthora sojae Avirulence Locus Avr3c Encodes a Multi-Copy RXLR Effector with Sequence Polymorphisms among Pathogen Strains

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 F2 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.

Oleoresinosis in Grand Fir (Abies grandis) Saplings and Mature Trees (Modulation of this Wound Response by Light and Water Stresses).

The stem content of diterpene resin acids (rosin) increases dramatically following wounding of grand fir (Abies grandis) saplings, but the level of monoterpene olefins (turpentine) in the stem decreases following injury, in spite of a significant increase in monoterpene cyclase (synthase) activity. However, this observation was explained when rapid evaporative losses of the volatile monoterpenes from the wound site was demonstrated by trapping experiments, a finding consistent with a role of turpentine as a solvent for the mobilization and deposition of rosin to seal the injury. Mature forest trees responded to stem wounding by the enhancement of monoterpene cyclization capacity in a manner similar to 2-year-old grand fir saplings raised in the greenhouse. Light and water stresses greatly reduced the constitutive level of monoterpene cyclase activity and abolished the wound-induced response. The diminution in monoterpene biosynthetic capacity was correlated with a dramatic decrease in cyclase protein as demonstrated by immunoblotting. Relief of stress conditions resulted in the restoration of cyclase activity (both constitutive and wound induced) to control levels. The results of these experiments indicate that grand fir saplings are a suitable model for studies of the regulation of defensive oleoresinosis in conifers.