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Fusarium graminearum is a causal agent of Fusarium head blight (FHB) and a deoxynivalenol (DON) producer. In this study, OSP24 is identified as an important virulence factor in systematic characterization of the 50 orphan secreted protein (OSP) genes of F. graminearum. Although dispensable for growth and initial penetration, OSP24 is important for infectious growth in wheat rachis tissues. OSP24 is specifically expressed during pathogenesis and its transient expression suppresses BAX- or INF1-induced cell death. Osp24 is translocated into plant cells and two of its 8 cysteine-residues are required for its function. Wheat SNF1-related kinase TaSnRK1α is identified as an Osp24-interacting protein and shows to be important for FHB resistance in TaSnRK1α-overexpressing or silencing transgenic plants. Osp24 accelerates the degradation of TaSnRK1α by facilitating its association with the ubiquitin-26S proteasome. Interestingly, TaSnRK1α also interacts with TaFROG, an orphan wheat protein induced by DON. TaFROG competes against Osp24 for binding with the same region of TaSnRKα and protects it from degradation. Overexpression of TaFROG stabilizes TaSnRK1α and increases FHB resistance. Taken together, Osp24 functions as a cytoplasmic effector by competing against TaFROG for binding with TaSnRK1α, demonstrating the counteracting roles of orphan proteins of both host and fungal pathogens during their interactions., Fusarium graminearum is a major fungal pathogen of cereals. Here the authors show that F. graminearum secretes an effector, Osp24, that induces degradation of the wheat TaSnRK1α kinase to promote disease while an orphan wheat protein, TaFROG1, can compete with Osp24 for binding to TaSnRK1α and protect it from degradation

The cAMP-PKA and MAP kinase pathways are essential for plant infection in the wheat head blight fungus Fusarium graminearum. To identify upstream receptors of these well-conserved signalling pathways, we systematically characterized the 105 G-protein-coupled receptor (GPCR) genes. Although none were required for vegetative growth, five GPCR genes (GIV1-GIV5) significantly upregulated during plant infection were important for virulence. The giv1 mutant was defective in the formation of specialized infection structures known as infection cushions, which was suppressed by application of exogenous cAMP and dominant active FST7 MEK kinase. GIV1 was important for the stimulation of PKA and Gpmk1 MAP kinase by compounds in wheat spikelets. GIV2 and GIV3 were important for infectious growth after penetration. Invasive hyphae of the giv2 mutant were defective in cell-to-cell spreading and mainly grew intercellularly in rachis tissues. Interestingly, the GIV2-GIV5 genes form a phylogenetic cluster with GIV6, which had overlapping functions with GIV5 during pathogenesis. Furthermore, the GIV2-GIV6 cluster is part of a 22-member subfamily of GPCRs, with many of them having in planta-specific upregulation and a common promoter element; however, only three subfamily members are conserved in other fungi. Taken together, F. graminearum has an expanded subfamily of infection-related GPCRs for regulating various infection processes.

Transcriptional plasticity enables oomycetes to rapidly adapt to environmental challenges including emerging host resistance. For example, the soybean pathogen Phytophthora sojae can overcome resistance conferred by the host resistance gene Rps1b through natural silencing of its corresponding effector gene, Avr1b-1. With the Phytophthora CRISPR/Cas9 genome editing system, it is possible to generate site-specific knock-out (KO) and knock-in (KI) mutants and to investigate the biological functions of target genes. In this study, the Avr1b-1 gene was deleted from the P. sojae genome using a homology-directed recombination strategy that replaced Avr1b-1 with a gene encoding the fluorescent protein mCherry. As expected, all selected KO transformants gained virulence on Rps1b plants, while infection of plants lacking Rps1b was not compromised. When a sgRNA-resistant version of Avr1b-1 was reintroduced into the Avr1b-1 locus of an Avr1b KO transformant, KI transformants with a well-transcribed Avr1b-1 gene were unable to infect Rps1b-containing soybeans. However, loss of expression of the incoming Avr1b-1 gene was frequently observed in KI transformants, which resulted in these transformants readily infecting Rps1b soybeans. A similar variability in the expression levels of the incoming gene was observed with AVI- or mCherry-tagged Avr1b-1 constructs. Our results suggest that Avr1b-1 may be unusually susceptible to transcriptional variation.

A cascade formed by phosphorylation events of mitogen‐activated protein kinases (MAPKs) takes part in plant stress responses. However, the roles of these MAPKs in resistance of potato (Solanum tuberosum) against Phytophthora pathogens is not well studied. Our previous work showed that a Phytophthora infestans RXLR effector targets and stabilizes the negative regulator of MAPK kinase 1 of potato (StMKK1). Because in Arabidopsis thaliana the AtMPK4 is the downstream phosphorylation target of AtMKK1, we performed a phylogenetic analysis and found that potato StMPK4/6/7 are closely related and are orthologs of AtMPK4/5/11/12. Overexpression of StMPK4/7 enhances plant resistance to P. infestans and P. parasitica. Yeast two‐hybrid analysis revealed that StMPK7 interacts with StMKK1, and StMPK7 is phosphorylated on flg22 treatment and by expressing constitutively active StMKK1 (CA‐StMKK1), indicating that StMPK7 is a direct downstream signalling partner of StMKK1. Overexpression of StMPK7 in potato enhances potato resistance to P. infestans. Constitutively active StMPK7 (CA‐StMPK7; StMPK7D198G, E202A) was found to promote immunity to Phytophthora pathogens and to trigger host cell death when overexpressed in Nicotiana benthamiana leaves. Cell death triggered by CA‐StMPK7 is SGT1/RAR1‐dependent. Furthermore, cell death triggered by CA‐StMPK7 is suppressed on coexpression with the salicylate hydroxylase NahG, and StMPK7 activation promotes salicylic acid (SA)‐responsive gene expression. We conclude that potato StMPK7 is a downstream signalling component of the phosphorelay cascade involving StMKK1 and StMPK7 plays a role in immunity to Phytophthora pathogens via an SA‐dependent signalling pathway., Potato StMPK7 is a downstream signalling component of StMKK1 and it enhances plant resistance to Phytophthora pathogens by activating the salicylic acid‐mediated signalling pathway.

Background The production of cereal crops is frequently affected by diseases caused by Fusarium graminearum and Magnaporthe oryzae, two devastating fungal pathogens. To improve crop resistance, many studies have focused on understanding the mechanisms of host defense against these two fungi individually. However, our knowledge of the common and different host defenses against these pathogens is very limited. Results In this study, we employed Brachypodium distachyon as a model for cereal crops and performed comparative transcriptomics to study the dynamics of host gene expression at different infection stages. We found that infection with either F. graminearum or M. oryzae triggered massive transcriptomic reprogramming in the diseased tissues. Numerous defense-related genes were induced with dynamic changes during the time course of infection, including genes that function in pattern detection, MAPK cascade, phytohormone signaling, transcription, protein degradation, and secondary metabolism. In particular, the expression of jasmonic acid signaling genes and proteasome component genes were likely specifically inhibited or manipulated upon infection by F. graminearum. Conclusions Our analysis showed that, although the affected host pathways are similar, their expression programs and regulations are distinct during infection by F. graminearum and M. oryzae. The results provide valuable insight into the interactions between B. distachyon and two important cereal pathogens.

Utilization of disease resistance components from wild potatoes is a promising and sustainable approach to control Phytophthora blight. Here, we combined avirulence (Avr) genes screen with RNA-seq analysis to discover the potential mechanism of resistance in Mexican wild potato species, Solanum pinnatisectum. Histological characterization displayed that hyphal expansion was significantly restricted in epidermal cells and mesophyll cell death was predominant, indicating that a typical defense response was initiated in S. pinnatisectum. Inoculation of S. pinnatisectum with diverse Phytophthora infestans isolates showed distinct resistance patterns, suggesting that S. pinnatisectum has complex genetic resistance to most of the prevalent races of P. infestans in northwestern China. Further analysis by Avr gene screens and comparative transcriptomic profiling revealed the presence and upregulation of multiple plant NBS-LRR genes corresponding to biotic stresses. Six NBS-LRR alleles of R1, R2, R3a, R3b, R4, and Rpi-smira2 were detected, and over 60% of the 112 detected NLR proteins were significantly induced in S. pinnatisectum. On the contrary, despite the expression of the Rpi-blb1, Rpi-vnt1, and Rpi-smira1 alleles, fewer NLR proteins were expressed in susceptible Solanum cardophyllum. Thus, the enriched NLR genes in S. pinnatisectum make it an ideal genetic resource for the discovery and deployment of resistance genes for potato breeding.

Utilization of disease resistance components from wild potatoes is a promising and sustainable approach to control Phytophthora blight. Here, we combined avirulence (Avr) genes screen with RNA-seq analysis to discover the potential mechanism of resistance in Mexican wild potato species, Solanum pinnatisectum. Histological characterization displayed that hyphal expansion was significantly restricted in epidermal cells and mesophyll cell death was predominant, indicating that a typical defense response was initiated in S. pinnatisectum. Inoculation of S. pinnatisectum with diverse Phytophthora infestans isolates showed distinct resistance patterns, suggesting that S. pinnatisectum has complex genetic resistance to most of the prevalent races of P. infestans in northwestern China. Further analysis by Avr gene screens and comparative transcriptomic profiling revealed the presence and upregulation of multiple plant NBS-LRR genes corresponding to biotic stresses. Six NBS-LRR alleles of R1, R2, R3a, R3b, R4, and Rpi-smira2 were detected, and over 60% of the 112 detected NLR proteins were significantly induced in S. pinnatisectum. On the contrary, despite the expression of the Rpi-blb1, Rpi-vnt1, and Rpi-smira1 alleles, fewer NLR proteins were expressed in susceptible Solanum cardophyllum. Thus, the enriched NLR genes in S. pinnatisectum make it an ideal genetic resource for the discovery and deployment of resistance genes for potato breeding.

Abscisic acid (ABA) signaling is a vital physiological step that is used by many land plants to fight against environmental stress. As components of the linear ABA signaling pathway, clade A protein phosphatases type 2C (PP2C-As) are mainly inhibited by PYRABACTIN RESISTANCE1/PYR1-LIKE/REGULATORY COMPONENTS OF ABA RECEPTORS (PYLs)-type receptors upon their binding to ABA. Here, we show that the genome of Brassica rapa encodes 14 putative clade A PP2C-like proteins (BrPP2C-As). Two of these BrPP2C-As, Bra025964 and Bra016595, show high similarity to the HAB2 (Homology to ABI2) protein in Arabidopsis. RNAseq data reveal that nearly all BrPP2C-As, like BrHAB2a (Bra025964) and BrHAB2b (Bra016595), were highly expressed in at least one tissue. Overexpression of BrHAB2a conferred ABA insensitivity to Arabidopsis thaliana seedlings. Furthermore, the phosphatase activity of BrHAB2a could be inhibited by AtPYL1 or BrPYL1 in the presence of ABA. Overall, these results suggest that BrHAB2a is a functional PP2C-A like protein phosphotase and a key component of ABA signaling in Brassica rapa.

Trichothecene mycotoxins, such as deoxynivalenol (DON) produced by the fungal pathogen, Fusarium graminearum, are not only important for plant infection but are also harmful to human and animal health. Trichothecene targets the ribosomal protein Rpl3 that is conserved in eukaryotes. Hence, a self-defense mechanism must exist in DON-producing fungi. It is reported that TRI (trichothecene biosynthesis) 101 and TRI12 are two genes responsible for self-defense against trichothecene toxins in Fusarium. In this study, however, we found that simultaneous disruption of TRI101 and TRI12 has no obvious influence on DON resistance upon exogenous DON treatment in F. graminearum, suggesting that other mechanisms may be involved in self-defense. By using RNA-seq, we identified 253 genes specifically induced in DON-treated cultures compared with samples from cultures treated or untreated with cycloheximide, a commonly used inhibitor of eukaryotic protein synthesis. We found that transporter genes are significantly enriched in this group of DON-induced genes. Of those genes, 15 encode major facilitator superfamily transporters likely involved in mycotoxin efflux. Significantly, we found that genes involved in the metabolism of gamma-aminobutyric acid (GABA), a known inducer of DON production in F. graminearum, are significantly enriched among the DON-induced genes. The GABA biosynthesis gene PROLINE UTILIZATION 2-2 (PUT2-2) is downregulated, while GABA degradation genes are upregulated at least twofold upon treatment with DON, resulting in decreased levels of GABA. Taken together, our results suggest that transporters influencing DON efflux are important for self-defense and that GABA mediates the balance of DON production and self-defense in F. graminearum.

Trichothecene mycotoxins, such as deoxynivalenol (DON) produced by the fungal pathogen, Fusarium graminearum, are not only important for plant infection but are also harmful to human and animal health. Trichothecene targets the ribosomal protein Rpl3 that is conserved in eukaryotes. Hence, a self-defense mechanism must exist in DON-producing fungi. It is reported that TRI (trichothecene biosynthesis) 101 and TRI12 are two genes responsible for self-defense against trichothecene toxins in Fusarium. In this study, however, we found that simultaneous disruption of TRI101 and TRI12 has no obvious influence on DON resistance upon exogenous DON treatment in F. graminearum, suggesting that other mechanisms may be involved in self-defense. By using RNA-seq, we identified 253 genes specifically induced in DON-treated cultures compared with samples from cultures treated or untreated with cycloheximide, a commonly used inhibitor of eukaryotic protein synthesis. We found that transporter genes are significantly enriched in this group of DON-induced genes. Of those genes, 15 encode major facilitator superfamily transporters likely involved in mycotoxin efflux. Significantly, we found that genes involved in the metabolism of gamma-aminobutyric acid (GABA), a known inducer of DON production in F. graminearum, are significantly enriched among the DON-induced genes. The GABA biosynthesis gene PROLINE UTILIZATION 2-2 (PUT2-2) is downregulated, while GABA degradation genes are upregulated at least twofold upon treatment with DON, resulting in decreased levels of GABA. Taken together, our results suggest that transporters influencing DON efflux are important for self-defense and that GABA mediates the balance of DON production and self-defense in F. graminearum.

Late blight caused by Phytophthora infestans is the most devastating disease of potato worldwide. To understand the P. infestans population structure and dynamics in northwestern China, 959 single-lesion isolates were purified in three consecutive years (2009–2011) and were characterized for mating type, pathotype, mtDNA haplotype and molecular variation at eight SSR loci. The results showed that the distribution of mating types changed significantly over years, with self-fertile isolates dominant in 2010 and 2011. SSR genotyping distinguished 959 isolates into 151 genotypes, and association analysis indicated that P. infestans populations in 2010 and 2011 were strictly asexual while in 2009 they showed signs of sexual reproduction. Population analysis showed that the majority of genetic variation was within P. infestans populations. Isolates sharing identical SSR genotypes were detected in distant regions, indicating that migration of P. infestans could have occurred between regions. Pathogenicity assays on a set of potato differential lines containing R1 to R11 resistance genes detected four pathotypes from 74 selected isolates, with the pathotype virulent against all 11 R genes being dominant. Three mtDNA haplotypes (Ia, IIa, IIb) were detected with Ia being dominant among 507 isolates examined. Phylogenetic analysis indicated that P. infestans populations in northwestern China are distant from European lineages including 13-A2 (blue-13) at the time of this survey. The results have implications for the trade of healthy seed tubers as a means of managing late blight.

Fusarium head blight, caused by Fusarium graminearum, is one of the most severe diseases on wheat and barley worldwide. Although the genomic data of several strains were published, the intragenomic variation of F. graminearum was not well characterized. Here, we sequenced three Chinese strains and conducted genome-wide comparisons. Our data revealed that all the sequenced strains were distinct from each other and over 350 genes were functionally lost in each of them. Variants of each strain were unevenly distributed in a highly conserved pattern along the chromosomes, resulting in a conserved two-speed genome. The fast subgenome has a lower GC content, shorter gene length, and higher variation of exon numbers than the slow subgenome. Genes related to interaction and pathogenicity, under positive selection, and up-regulated in planta were all significantly enriched in the fast subgenome. Furthermore, we found that the fast subgenome coincided with facultative heterochromatin regions that were repressed in vegetative stage but activated during infection as measured by RNA-seq and ChIP-seq data, suggesting that the fast subgenome is epigenetically regulated. Taken together, our data demonstrated that F. graminearum has a highly conserved two-speed genome and the fast subgenome responsible for adaption and infection is under the control of heterochromatin.

Very long chain fatty acids (VLCFAs) are essential lipid components in many plants. 3-Ketoacyl-CoA synthase (KCS) catalyzes the condensation reaction to form 3-ketoacyl-CoA in VLCFA synthesis. AtELO4 has been reported to be involved in VLCFA synthesis, functioning as a KCS in Arabidopsis. However, no studies on other three AtELO members have been reported. Here, we initially found by real-time PCR in Arabidopsis thaliana (L.) Heynh. that AtELO1, AtELO3, and AtELO4 displayed characteristic expression patterns, but AtELO2 was nearly expressed in any organ. Then the transient expression of ELO-like-eGFP fusions in Arabidopsis green leaf protoplasts showed that AtELO1, AtELO3, and AtELO4 were localized in the endoplasmic reticulum (ER), where VLCFA synthesis took place. Finally, we found that the contents of all fatty acids were decreased by 10–20% in seeds of atelo1 T-DNA insertion mutants. In seeds of Pro35S:AtELO1 plants, the levels of all remaining components, except C20:0 and C20:3, were significantly increased. Taken together, our study revealed biological functions of AtELO members and might lay the foundation for further genetic manipulations to generate oil crops with the high oil content.

Although peanut (Arachis hypogaea L.) is a relatively drought-tolerant crop, its productivity and quality is adversely affected by drought stress. The identification of novel genes, the analysis of gene expression patterns in response to dehydration stress, and the evaluation of their potential functions in stress adaptation will provide the basis for effective engineering strategies to improve crop drought tolerance. However, little is known about stress tolerance of peanut. To investigate genes that are involved in the response to drought stress in peanut, a survey of genes responsive to drought stress was done by cDNA macroarray analysis, and about 277 unigenes were selected, including genes related among others to stress response and transcription factors. Totally, it was found that about 21 clones could respond to dehydration. The expression patterns of these 21 candidate drought-responsive genes and another 30 selected non-responsive genes were further analyzed by real-time PCR, and then we found another responsive gene P3617. Among these genes, 18 genes were up-regulated, and 4 genes were down-regulated by drought. The functional classification indicated that these genes belonged to gene families encoding stress-responsive proteins, transcription factors, auxin-responsive protein, Na+/H+ transporter SOS1, and chloroplast photosynthetic oxygen-evolving protein subunit of peanut. Collectively, the data presented in this study will facilitate the understanding of molecular mechanism of the peanut response to drought stress and also provide the potential strategies for effective genetic engineering to improve peanut stress tolerance.

Fusarium head blight, caused by Fusarium graminearum, is one of the most severe diseases on wheat and barley worldwide. Although the genomic data of several strains were published, the intragenomic variation of F. graminearum was not well characterized. Here, we sequenced three Chinese strains and conducted genome-wide comparisons. Our data revealed that all the sequenced strains were distinct from each other and over 350 genes were functionally lost in each of them. Variants of each strain were unevenly distributed in a highly conserved pattern along the chromosomes, resulting in a conserved two-speed genome. The fast subgenome has a lower GC content, shorter gene length, and higher variation of exon numbers than the slow subgenome. Genes related to interaction and pathogenicity, under positive selection, and up-regulated in planta were all significantly enriched in the fast subgenome. Furthermore, we found that the fast subgenome coincided with facultative heterochromatin regions that were repressed in vegetative stage but activated during infection as measured by RNA-seq and ChIP-seq data, suggesting that the fast subgenome is epigenetically regulated. Taken together, our data demonstrated that F. graminearum has a highly conserved two-speed genome and the fast subgenome responsible for adaption and infection is under the control of heterochromatin.

Yeasts and filamentous fungi do not have adenosine deaminase acting on RNA (ADAR) orthologs and are believed to lack A-to-I RNA editing, which is the most prevalent editing of mRNA in animals. However, during this study with the PUK1 (FGRRES_01058) pseudokinase gene important for sexual reproduction in Fusarium graminearum, we found that two tandem stop codons, UA1831GUA1834G, in its kinase domain were changed to UG1831GUG1834G by RNA editing in perithecia. To confirm A-to-I editing of PUK1 transcripts, strand-specific RNA-seq data were generated with RNA isolated from conidia, hyphae, and perithecia. PUK1 was almost specifically expressed in perithecia, and 90% of transcripts were edited to UG1831GUG1834G. Genome-wide analysis identified 26,056 perithecium-specific A-to-I editing sites. Unlike those in animals, 70.5% of A-to-I editing sites in F. graminearum occur in coding regions, and more than two-thirds of them result in amino acid changes, including editing of 69 PUK1-like pseudogenes with stop codons in ORFs. PUK1 orthologs and other pseudogenes also displayed stage-specific expression and editing in Neurospora crassa and F. verticillioides. Furthermore, F. graminearum differs from animals in the sequence preference and structure selectivity of A-to-I editing sites. Whereas A's embedded in RNA stems are targeted by ADARs, RNA editing in F. graminearum preferentially targets A's in hairpin loops, which is similar to the anticodon loop of tRNA targeted by adenosine deaminases acting on tRNA (ADATs). Overall, our results showed that A-to-I RNA editing occurs specifically during sexual reproduction and mainly in the coding regions in filamentous ascomycetes, involving adenosine deamination mechanisms distinct from metazoan ADARs.

An Acidovorax citrulli–cucumber pathosystem was established through which A. citrulli mutants with altered pathogenicity, generated by transposon mutagenesis, were identified on cucumber cotyledons. The A. citrulli group I strain FC440 was shown to grow faster in cucumber leaf tissues than a group II strain and was used for Tn5 transposon mutagenesis. A total of 2100 Tn5 insertional mutants were generated, and analysis of the mutant library showed that the transposon insertions were single, independent and stable. A conserved non-flagellar type III secretion system (NF-T3SS) ATPase gene hrcN was identified and confirmed to be essential for pathogenicity and functionality of NF-T3SS in A. citrulli. Comparative sequence analysis of the HrcN protein and its homologues in other representative bacterial plant pathogens revealed that the NF-T3SS of A. citrulli is close to that of Ralstonia solanacearum and Xanthomonas campestris, but distant from that of Pseudomonas syringae and Erwinia amylovora. The generated Tn5 insertional mutant collection is valuable for identification of genes required for A. citrulli pathogenesis, and the established A. citrulli–cucumber pathosystem will facilitate an improved understanding of A. citrulli biology and pathology.

Protein disulfide isomerase (PDI) is a ubiquitous and multifunction enzyme belonging to the thioredoxin (TRX) superfamily, which can reduce, oxidize, and catalyze dithiol–disulfide exchange reactions. Other than performing housekeeping functions in helping to maintain proteins in a more stable conformation, there is some evidence to indicate that PDI is involved in pathogen infection processes. In a high-throughput screening for necrosis-inducing factors by Agrobacterium tumefaciens-mediated transient expression assay, a typical PDI gene from Phytophthora parasitica (PpPDI1) was identified and confirmed to induce strong cell death in Nicotiana benthamiana leaves. PpPDI1 is conserved in eukaryotes but predicted to be a secreted protein. Deletion mutant analyses showed that the first CGHC motif in the active domain of PpPDI1 is essential for inducing cell death. Using P. parasitica transformation method, the silencing efficiency was found to be very low, suggesting that PpPDI1 is essential for the pathogen. Translational fusion to the enhanced green fluorescent protein (EGFP) in stable P. parasitica transformants showed that PpPDI1 is associated with haustoria-like structures during pathogen infection. Furthermore, the PpPDI1-EGFP-expressing transformants increase the number of haustoria-like structures and exhibit enhanced virulence to N. benthamiana. These results indicate that PpPDI1 might be a virulence factor of P. parasitica and contributes to plant infection.

The interaction between Arabidopsis thaliana and the oomycete pathogen Phytophthora parasitica emerges as a model for exploring the molecular basis and evolution of recognition and host defense. Phenotypic variation and genetic analysis is essential to dissect the underlying mechanisms in plant-oomycete interaction. In this study, the reaction phenotypes of 28 A. thaliana accessions to P. parasitica strain Pp016 were examined using detached leaf infection assay. The results showed the presence of four distinct groups based on host response and disease development. Of all the accessions examined, Zurich (Zu-1) is highly resistant to P. parasitica. Microscopic characterization showed that rapid and severe hypersensitive response (HR) at the primary infection epidermal cells is associated with disease resistance. Furthermore, Zu-1 is resistant to a set of 20 diverse P. parasitica strains, which were collected from different host plants and exhibited differential specificities on a set of tobacco cultivars. However, Zu-1 is susceptible to P. parasitica when the root is inoculated, suggesting differential expression of associated resistance genes in the root and foliar tissues. Genetic analysis by crossing Zu-1 and the susceptible accession Landsberg (Ler) showed that the resistance in Zu-1 to P. parasitica is semi-dominant, as shown by infection assays of F1 progenies, and is likely conferred by a single locus, defined as RPPA1Zu-1 (for Resistance to Phytophthora parasitica 1), as shown by analysis of F2 segregating populations. By employing specific-locus amplified fragment sequencing (SLAF-seq) strategy to identify molecular markers potentially linked to the locus, the strongest associated region was determined to be located between 7.1-11.2 Mb in chromosome IV. The future cloning of RPPA1Zu-1 locus will facilitate improved understanding of plant broad-spectrum disease resistance to oomycete pathogens.

In cells, metallochaperones are important proteins that safely transport metal ions. Heavy metal-associated isoprenylated plant proteins (HIPPs) are metallochaperones that contain a metal binding domain and a CaaX isoprenylation motif at the carboxy-terminal end. To investigate the roles of wheat heavy metal-associated isoprenylated plant protein (TaHIPP) genes in plant development and in stress responses, we isolated cDNA encoding the wheat TaHIPP1 gene, which contains a heavy metal-associated domain, nuclear localisation signals and an isoprenylation motif (CaaX motif). Quantitative real-time PCR analysis indicated that the TaHIPP1 gene was differentially expressed under biotic and abiotic stresses. Specifically, TaHIPP1 expression was up-regulated by ABA exposure or wounding. Additionally, TaHIPP1 over-expression in yeast (Schizosaccharomyces pombe) significantly increased the cell growth rate under Cu(2+) and high salinity stresses. The nuclear localisation of the protein was confirmed with confocal laser scanning microscopy of epidermal onion cells after particle bombardment with chimeric TaHIPP1-GFP constructs. In addition, TaHIPP1 was shown to enhance the susceptibility of wheat to Pst as determined by virus-induced gene silencing. These data indicate that TaHIPP1 is an important component in defence signalling pathways and may play a crucial role in the defence response of wheat to biotic and certain abiotic stresses.

bZIP (basic leucine zipper) transcription factors coordinate plant growth and development and control responses to environmental stimuli. The genome of Chinese cabbage (Brassica rapa) encodes 136 putative bZIP transcription factors. The bZIP transcription factors in Brassica rapa (BrbZIP) are classified into 10 subfamilies. Phylogenetic relationship analysis reveals that subfamily A consists of 23 BrbZIPs. Two BrbZIPs within subfamily A, Bra005287 and Bra017251, display high similarity to ABI5 (ABA Insensitive 5). Expression of subfamily A BrbZIPs, like BrABI5a (Bra005287/BrbZIP14) and BrABI5b (Bra017251/BrbZIP13), are significantly induced by the plant hormone ABA. Subcellular localization assay reveal that both BrABI5a and BrABI5b have a nuclear localization. BrABI5a and BrABI5b could directly stimulate ABA Responsive Element-driven HIS (a HIS3 reporter gene, which confers His prototrophy) or LUC (LUCIFERASE) expression in yeast and Arabidopsis protoplast. Deletion of the bZIP motif abolished BrABI5a and BrABI5b transcriptional activity. The ABA insensitive phenotype of Arabidopsis abi5-1 is completely suppressed in transgenic lines expressing BrABI5a or BrABI5b. Overall, these results suggest that ABI5 orthologs, BrABI5a and BrABI5b, have key roles in ABA signalling in Chinese cabbage.

BackgroundEffector proteins of biotrophic plant pathogenic fungi and oomycetes are delivered into host cells and play important roles in both disease development and disease resistance response. How obligate fungal pathogen effectors enter host cells is poorly understood. The Ps87 gene of Puccinia striiformis encodes a protein that is conserved in diverse fungal pathogens. Ps87 homologs from a clade containing rust fungi are predicted to be secreted. The aim of this study is to test whether Ps87 may act as an effector during Puccinia striiformis infection.Methodology/principal findingsYeast signal sequence trap assay showed that the rust protein Ps87 could be secreted from yeast cells, but a homolog from Magnaporthe oryzae that was not predicted to be secreted, could not. Cell re-entry and protein uptake assays showed that a region of Ps87 containing a conserved RXLR-like motif [K/R]RLTG was confirmed to be capable of delivering oomycete effector Avr1b into soybean leaf cells and carrying GFP into soybean root cells. Mutations in the Ps87 motif (KRLTG) abolished the protein translocation ability.Conclusions/significanceThe results suggest that Ps87 and its secreted homologs could utilize similar protein translocation machinery as those of oomycete and other fungal pathogens. Ps87 did not show direct suppression activity on plant defense responses. These results suggest Ps87 may represent an "emerging effector" that has recently acquired the ability to enter plant cells but has not yet acquired the ability to alter host physiology.

Species of the oomycete genus Phytophthora are destructive pathogens, causing extensive losses in agricultural crops and natural ecosystems. A potential disease control approach is the application of RNA silencing technology which has proven to be effective in improving plant resistance against a wide range of pests including parasitic plants, nematodes, insects and fungi. In this study, we tested the potential application of RNA silencing in improving plant disease resistance against oomycete pathogens. The endogenous P. parasitica gene PnPMA1 and the reporter gene GFP were used to evaluate the potential application of host induced gene silencing (HIGS). The GFP-expressing P. parasitica efficiently colonized Arabidopsis thaliana lines stably expressing GFP dsRNA and showed no obvious decrease in GFP signal intensity. Quantitative RT-PCR analyses showed no significant reductions in the abundance of GFP and PnPMA1 transcripts in P. parasitica during colonization of A. thaliana lines stably expressing GFP and PnPMA1 dsRNAs, respectively. Neither GFP siRNAs nor PnPMA1 siRNAs produced by transgenic plants were detected in P. parasitica re-isolated from infected tissues by Northern blot analyses. Phenotypic characterization of zoospores released from infected plant roots expressing PnPMA1 dsRNA showed no motility changes compared with those from wild-type plants. Similar results were obtained by analysis of zoospores released from sporulating hyphae of P. parasitica re-isolated from PnPMA1 dsRNA-expressing plant roots. Thus, the ectopic expression of dsRNA sequences in the host plant is not sufficient to initiate silencing of homologous genes in the colonizing oomycete pathogen, and this may be due to a number of different reasons including the absence of genetic machinery required for uptake of silencing signals in particular dsRNAs which are essential for environmental RNA silencing.

Oomycete pathogens cause severe damage to a wide range of agriculturally important crops and natural ecosystems. They represent a unique group of plant pathogens that are evolutionarily distant from true fungi. In this study, we established a new plant–oomycete pathosystem in which the broad host range pathogen Phytophthora parasitica was demonstrated to be capable of interacting compatibly with the model plant Arabidopsis thaliana. Water‐soaked lesions developed on leaves within 3 days and numerous sporangia formed within 5 days post‐inoculation of P. parasitica zoospores. Cytological characterization showed that P. parasitica developed appressoria‐like swellings and penetrated epidermal cells directly and preferably at the junction between anticlinal host cell walls. Multiple haustoria‐like structures formed in both epidermal cells and mesophyll cells 1 day post‐inoculation of zoospores. Pathogenicity assays of 25 A. thaliana ecotypes with six P. parasitica strains indicated the presence of a natural variation in host specificity between A. thaliana and P. parasitica. Most ecotypes were highly susceptible to P. parasitica strains Pp014, Pp016 and Pp025, but resistant to strains Pp008 and Pp009, with the frequent appearance of cell wall deposition and active defence response‐based cell necrosis. Gene expression and comparative transcriptomic analysis further confirmed the compatible interaction by the identification of up‐regulated genes in A. thaliana which were characteristic of biotic stress. The established A. thaliana–P. parasitica pathosystem expands the model systems investigating oomycete–plant interactions, and will facilitate a full understanding of Phytophthora biology and pathology.