Yuanchao Wang

Signatures of adaptation to obligate biotrophy in the Hyaloperonospora arabidopsidis genome.

From Blight to Powdery Mildew Pathogenic effects of microbes on plants have widespread consequences. Witness, for example, the cultural upheavals driven by potato blight in the 1800s. A variety of microbial pathogens continue to afflict crop plants today, driving both loss of yield and incurring the increased costs of control mechanisms. Now, four reports analyze microbial genomes in order to understand better how plant pathogens function (see the Perspective by Dodds). Raffaele et al. (p. 1540) describe how the genome of the potato blight pathogen accommodates transfer to different hosts. Spanu et al. (p. 1543) analyze what it takes to be an obligate biotroph in barley powdery mildew, and Baxter et al. (p. 1549) ask a similar question for a natural pathogen of Arabidopsis. Schirawski et al. (p. 1546) compared genomes of maize pathogens to identify virulence determinants. Better knowledge of what in a genome makes a pathogen efficient and deadly is likely to be useful for improving agricultural crop management and breeding. A group of papers analyzes pathogen genomes to find the roots of virulence, opportunism, and life-style determinants. Many oomycete and fungal plant pathogens are obligate biotrophs, which extract nutrients only from living plant tissue and cannot grow apart from their hosts. Although these pathogens cause substantial crop losses, little is known about the molecular basis or evolution of obligate biotrophy. Here, we report the genome sequence of the oomycete Hyaloperonospora arabidopsidis (Hpa), an obligate biotroph and natural pathogen of Arabidopsis thaliana. In comparison with genomes of related, hemibiotrophic Phytophthora species, the Hpa genome exhibits dramatic reductions in genes encoding (i) RXLR effectors and other secreted pathogenicity proteins, (ii) enzymes for assimilation of inorganic nitrogen and sulfur, and (iii) proteins associated with zoospore formation and motility. These attributes comprise a genomic signature of evolution toward obligate biotrophy.

Conserved C-Terminal Motifs Required for Avirulence and Suppression of Cell Death by Phytophthora sojae effector Avr1b

The sequenced genomes of oomycete plant pathogens contain large superfamilies of effector proteins containing the protein translocation motif RXLR-dEER. However, the contributions of these effectors to pathogenicity remain poorly understood. Here, we show that the Phytophthora sojae effector protein Avr1b can contribute positively to virulence and can suppress programmed cell death (PCD) triggered by the mouse BAX protein in yeast, soybean (Glycine max), and Nicotiana benthamiana cells. We identify three conserved motifs (K, W, and Y) in the C terminus of the Avr1b protein and show that mutations in the conserved residues of the W and Y motifs reduce or abolish the ability of Avr1b to suppress PCD and also abolish the avirulence interaction of Avr1b with the Rps1b resistance gene in soybean. W and Y motifs are present in at least half of the identified oomycete RXLR-dEER effector candidates, and we show that three of these candidates also suppress PCD in soybean. Together, these results indicate that the W and Y motifs are critical for the interaction of Avr1b with host plant target proteins and support the hypothesis that these motifs are critical for the functions of the very large number of predicted oomycete effectors that contain them.

Transcriptional Programming and Functional Interactions within the Phytophthora sojae RXLR Effector Repertoire

This study presents a broad functional survey of a large sample of candidate RXLR effectors in the oomycete pathogen of soybean (Phytophthora sojae). Suppression of plant defense, transcription patterns, and polymorphisms were assayed. Essential effectors and effector subsets with distinct expression patterns and defense suppression activities were identified. The genome of the soybean pathogen Phytophthora sojae contains nearly 400 genes encoding candidate effector proteins carrying the host cell entry motif RXLR-dEER. Here, we report a broad survey of the transcription, variation, and functions of a large sample of the P. sojae candidate effectors. Forty-five (12%) effector genes showed high levels of polymorphism among P. sojae isolates and significant evidence for positive selection. Of 169 effectors tested, most could suppress programmed cell death triggered by BAX, effectors, and/or the PAMP INF1, while several triggered cell death themselves. Among the most strongly expressed effectors, one immediate-early class was highly expressed even prior to infection and was further induced 2- to 10-fold following infection. A second early class, including several that triggered cell death, was weakly expressed prior to infection but induced 20- to 120-fold during the first 12 h of infection. The most strongly expressed immediate-early effectors could suppress the cell death triggered by several early effectors, and most early effectors could suppress INF1-triggered cell death, suggesting the two classes of effectors may target different functional branches of the defense response. In support of this hypothesis, misexpression of key immediate-early and early effectors severely reduced the virulence of P. sojae transformants.

The bZIP transcription factor MoAP1 mediates the oxidative stress response and is critical for pathogenicity of the rice blast fungus Magnaporthe oryzae.

Saccharomyces cerevisiae Yap1 protein is an AP1-like transcription factor involved in the regulation of the oxidative stress response. An ortholog of Yap1, MoAP1, was recently identified from the rice blast fungus Magnaporthe oryzae genome. We found that MoAP1 is highly expressed in conidia and during invasive hyphal growth. The Moap1 mutant was sensitive to H2O2, similar to S. cerevisiae yap1 mutants, and MoAP1 complemented Yap1 function in resistance to H2O2, albeit partially. The Moap1 mutant also exhibited various defects in aerial hyphal growth, mycelial branching, conidia formation, the production of extracellular peroxidases and laccases, and melanin pigmentation. Consequently, the Moap1 mutant was unable to infect the host plant. The MoAP1-eGFP fusion protein is localized inside the nucleus upon exposure to H2O2, suggesting that MoAP1 also functions as a redox sensor. Moreover, through RNA sequence analysis, many MoAP1-regulated genes were identified, including several novel ones that were also involved in pathogenicity. Disruption of respective MGG_01662 (MoAAT) and MGG_02531 (encoding hypothetical protein) genes did not result in any detectable changes in conidial germination and appressorium formation but reduced pathogenicity, whereas the mutant strains of MGG_01230 (MoSSADH) and MGG_15157 (MoACT) showed marketed reductions in aerial hyphal growth, mycelial branching, and loss of conidiation as well as pathogenicity, similar to the Moap1 mutant. Taken together, our studies identify MoAP1 as a positive transcription factor that regulates transcriptions of MGG_01662, MGG_02531, MGG_01230, and MGG_15157 that are important in the growth, development, and pathogenicity of M. oryzae.

Oomycete pathogens encode RNA silencing suppressors

Effectors are essential virulence proteins produced by a broad range of parasites, including viruses, bacteria, fungi, oomycetes, protozoa, insects and nematodes. Upon entry into host cells, pathogen effectors manipulate specific physiological processes or signaling pathways to subvert host immunity. Most effectors, especially those of eukaryotic pathogens, remain functionally uncharacterized. Here, we show that two effectors from the oomycete plant pathogen Phytophthora sojae suppress RNA silencing in plants by inhibiting the biogenesis of small RNAs. Ectopic expression of these Phytophthora suppressors of RNA silencing enhances plant susceptibility to both a virus and Phytophthora, showing that some eukaryotic pathogens have evolved virulence proteins that target host RNA silencing processes to promote infection. These findings identify RNA silencing suppression as a common strategy used by pathogens across kingdoms to cause disease and are consistent with RNA silencing having key roles in host defense.

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.

The Basic Leucine Zipper Transcription Factor Moatf1 Mediates Oxidative Stress Responses and Is Necessary for Full Virulence of the Rice Blast Fungus Magnaporthe oryzae

Magnaporthe oryzae is the causal agent of rice blast disease, leading to enormous losses of rice production. Here, we characterized a basic leucine zipper (bZIP) transcription factor, Moatf1, in M. oryzae, a homolog of Schizosaccharomyces pombe ATF/CREB that regulates the oxidative stress response. Moatf1 deletion caused retarded vegetative growth of mycelia, and the Moatf1 mutant exhibited higher sensitivity to hydrogen peroxide (H(2)O(2)) than did the wild-type strain. The mutant showed severely reduced activity of extracellular enzymes and transcription level of laccases and peroxidases and exhibited significantly reduced virulence on rice cultivar CO-39. On rice leaf sheath, most of the infectious hyphae of the mutant became swollen and displayed restricted growth in primary infected cells. Defense response was strongly activated in plants infected by the mutant. Diamino benzidine staining revealed an accumulation of H(2)O(2) around Moatf1 mutant appressoria and rice cells with Moatf1 hyphae that was absent in the wild type. Inhibition of the plant NADPH oxidase by diphenyleneiodonium prevented host-derived H(2)O(2) accumulation and restored infectious hyphal growth of the mutant in rice cells. Thus, we conclude that Moatf1 is necessary for full virulence of M. oryzae by regulating the transcription of laccases and peroxidases to impair reactive oxygen species-mediated plant defense.

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.

MgCRZ1, a transcription factor of Magnaporthe grisea, controls growth, development and is involved in full virulence.

Calcineurin, a conserved Ca(2+)/calmodulin-regulated protein phosphatase, is an important mediator of calcium-dependent signal transduction pathways in many organisms. In Saccharomyces cerevisiae, calcineurin positively regulates transcription in response to stress by dephosphorylating the transcription factor Crz1p. Here we describe the identification, cloning, and function of the gene encoding the Magnaporthe grisea CRZ1 homolog, MgCRZ1. Specifically, we demonstrated that MgCRZ1 partially complemented a yeast Deltacrz1 mutant and exhibited Ca(2+) and calcineurin activity-dependent cellular localization. Targeted disruption of MgCRZ1 resulted in hypersensitivity to Ca(2+). Compared with the wild-type Guy11 strain, the Deltacrz1 mutants formed significantly reduced numbers of conidia and a large portion of abnormal appressoria (>50%) that exhibited little or no melanin production. Lipid metabolism was delayed, and the level of turgor pressure within the appressoria declined, thereby notably attenuating mutant pathogenicity. We conclude that MgCRZ1 is essential for growth, development, and full virulence of M. grisea.