Establishment of a simple and efficient Agrobacterium-mediated transformation system for Phytophthora palmivora.
ABSTRACT: As an agriculturally important oomycete genus, Phytophthora contains a large number of destructive plant pathogens that severely threaten agricultural production and natural ecosystems. Among them is the broad host range pathogen P. palmivora, which infects many economically important plant species. An essential way to dissect their pathogenesis mechanisms is genetic modification of candidate genes, which requires effective transformation systems. Four methods were developed for transformation of Phytophthora spp., including PEG(polyethylene glycol)/CaCl2 mediated protoplast transformation, electroporation of zoospores, microprojectile bombardment and Agrobacterium-mediated transformation (AMT). Among them, AMT has many advantages over the other methods such as easy handling and mainly generating single-copy integration in the genome. An AMT method previously reported for P. infestans and P. palmivora has barely been used in oomycete research due to low success and low reproducibility.In this study, we report a simple and efficient AMT system for P. palmivora. Using this system, we were able to reproducibly generate over 40 transformants using zoospores collected from culture grown in a single 100 mm-diameter petri dish. The generated GFP transformants constitutively expressed GFP readily detectable using a fluorescence microscope. All of the transformants tested using Southern blot analysis contained a single-copy T-DNA insertion.This system is highly effective and reproducible for transformation of P. palmivora and expected to be adaptable for transformation of additional Phytophthora spp. and other oomycetes. Its establishment will greatly accelerate their functional genomic studies.
Project description:BACKGROUND:Oomycetes are pathogens of mammals, fish, insects and plants, and the potato late blight agent Phytophthora infestans and the oil palm and cocoa infecting pathogen Phytophthora palmivora cause economically impacting diseases on a wide range of crop plants. Increasing genomic and transcriptomic resources and recent advances in oomycete biology demand new strategies for genetic modification of oomycetes. Most oomycete transformation procedures rely on geneticin-based selection of transgenic strains. RESULTS:We established N-acetyltransferase AAC(3)-I as a gentamicin-based selectable marker for oomycete transformation without interference with existing geneticin resistance. Strains carrying gentamicin resistance are fully infectious in plants. We further demonstrate the usefulness of this new antibiotic selection to super-transform well-characterized, already fluorescently-labelled P. palmivora strains and provide a comprehensive protocol for maintenance and zoospore electro-transformation of Phytophthora strains to aid in plant-pathogen research. CONCLUSIONS:N-acetyltransferase AAC(3)-I is functional in Phytophthora oomycetes. In addition, the substrate specificity of the AAC(3)-I enzyme allows for re-transformation of geneticin-resistant strains. Our findings and resources widen the possibilities to study oomycete cell biology and plant-oomycete interactions.
Project description:Bud rot (BR) is the most devastating disease affecting oil palm (Elaeis guineensis) crops in Colombia. Its causal agent, Phytophthora palmivora, initiates the infection in immature oil palm leaflets producing necrotic lesions, followed by colonization of opportunistic necrotrophs, which increases disease damage. To improve the characterization of the disease, we transformed P. palmivora using Agrobacterium tumefaciens-mediated transformation (ATMT) to include the fluorescent proteins CFP-SKL (peroxisomal localization), eGFP and mRFP1 (cytoplasmic localization). The stability of some transformants was confirmed by Southern blot analysis and single zoospore cultures; additionally, virulence and in vitro growth were compared to the wild-type isolate to select transformants with the greatest resemblance to the WT isolate. GFP-tagged P. palmivora was useful to identify all of the infective structures that are commonly formed by hemibiotrophic oomycetes, including apoplastic colonization and haustorium formation. Finally, we detected cell death responses associated with immature oil palm tissues that showed reduced susceptibility to P. palmivora infection, indicating that these tissues could exhibit age-related resistance. The aim of this research is to improve the characterization of the initial disease stages and generate cell biology tools that may be useful for developing methodologies for early identification of oil palm materials resistant or susceptible to BR.
Project description:Phytophthora palmivora is a destructive oomycete plant pathogen with a wide host range. So far, little is known about the factors governing its infection structure development and pathogenicity. From the culture filtrate of a P. palmivora strain isolated from papaya, we identified a secreted glycoprotein of 15?kDa, designated as Ppal15kDa, using liquid chromatography tandem mass spectrometry. Two gene variants, Ppal15kDaA and Ppal15kDaB were amplified from a P. palmivora papaya isolate. Transient expression of both variants in Nicotiana benthamiana by agroinfiltration enhanced P. palmivora infection. Six Ppal15kDa mutants with diverse mutations were generated via CRISPR/Cas9-mediated gene editing. All mutants were compromised in infectivity on N. benthamiana and papaya. Two mutants with all Ppal15kDa copies mutated almost completely lost pathogenicity. The pathogenicity of the other four containing at least one wild-type copy of Ppal15kDa was compromised at varying levels. The mutants were also affected in development as they produced smaller sporangia, shorter germ tubes, and fewer appressoria. The affected levels in development corresponded to the levels of reduction in pathogenicity, suggesting that Ppal15kDa plays an important role in normal development of P. palmivora infection structures. Consistent with its role in infection structure development and pathogenicity, Ppal15kDa was found to be highly induced during appressorium formation. In addition, Ppal15kDa homologs are broadly present in Phytophthora spp., but none were characterized. Altogether, this study identified a novel component involved in development and pathogenicity of P. palmivora and possibly other Phytophthora spp. known to contain a Ppal15kDa homolog.
Project description:Phytophthora spp. is an oomycetes pathogen which causes serious damage to a wide range of crops. Bud rot disease of coconut palm, caused by P. palmivora, causes huge economic losses since it cannot be detected at an early stage. Utilizing dual RNA-sequencing (RNA-seq), we have simultaneously investigated the gene expression patterns in both, the infecting oomycete (P. palmivora) and infected host (coconut leaflets). Samples were collected at three time points viz., 12, 24 and 36 h, from both infected and uninfected (control) tissues and subjected to RNA-seq on an Illumina Hiseq™ 2500 sequencing platform. High quality reads obtained were subjected to mapping with corresponding reference genomes by using the HISAT2/ StringTie package. A total of 81,683 transcripts were generated against the coconut reference genome, while 9340 transcripts were generated against P. palmivora genome. Out of these, a total of 64,639 coconut transcripts and 9168 P. palmivora transcripts could be annotated using BLASTx. Gene ontology (GO) analysis, carried out using Blast2GO, resulted in 212,643 coconut and 30,736 P palmivora transcripts being functionally classified, with a single gene product described by numerous terms under the three classifications. The insights obtained could contribute to an understanding of pathogenesis of P. palmivora and inducible defense response of coconut leaves to P. palmivora.
Project description:Phytophthora palmivora is an oomycete that causes oil palm bud rot disease. To understand the molecular mechanisms of this disease, palm clones with contrasting responses (Ortet 34, resistant and Ortet 57, susceptible) were inoculated with P. palmivora, and RNAseq gene expression analysis was performed. The transcriptome was obtained by sequencing using Illumina HiSeq2500 technology during the asymptomatic phase (24, 72 and 120 hours postinfection, hpi). A simultaneous analysis of differentially expressed gene (DEG) profiles in palm and P. palmivora was carried out. Additionally, Gene Ontology (GO) and gene network analysis revealed differences in the transcriptional profile of the two ortets, where a high specificity of the pathogen to colonize the susceptible ortet was found. The transcriptional analysis provided an overview of the genes involved in the recognition and signaling of this pathosystem, where different transcription factors, phytohormones, proteins associated with cell wall hardening and nitrogen metabolism contribute to the resistance of oil palm to P. palmivora. This research provides a description of the molecular response of oil palm to P. palmivora, thus becoming an important source of molecular markers for the study of genotypes resistant to bud rot disease.
Project description:The expansion of plants onto land was a formative event that brought forth profound changes to the earth's geochemistry and biota. Filamentous eukaryotic microbes developed the ability to colonize plant tissues early during the evolution of land plants, as demonstrated by intimate, symbiosis-like associations in >400 million-year-old fossils. However, the degree to which filamentous microbes establish pathogenic interactions with early divergent land plants is unclear. Here, we demonstrate that the broad host-range oomycete pathogen Phytophthora palmivora colonizes liverworts, the earliest divergent land plant lineage. We show that P. palmivora establishes a complex tissue-specific interaction with Marchantia polymorpha, where it completes a full infection cycle within air chambers of the dorsal photosynthetic layer. Remarkably, P. palmivora invaginates M. polymorpha cells with haustoria-like structures that accumulate host cellular trafficking machinery and the membrane syntaxin MpSYP13B, but not the related MpSYP13A. Our results indicate that the intracellular accommodation of filamentous microbes is an ancient plant trait that is successfully exploited by pathogens like P. palmivora.
Project description:Here, we define the proteomic response of the early divergent liverwort Marchantia polymorpha during infection with the oomycete pathogen Phytophthora palmivora. We sampled whole liverwort thalli that were mock-inoculated (water) or infected with P. palmivora zoospores at 4 and 8 days post inoculation (dpi). This analysis revealed the protein profiles of liverworts during the biotrophic (4 dpi) and necrotrophic (8 dpi) stages of pathogen infection. In combination with additional omics datasets, our analyses reveal conserved aspects in the molecular response to pathogen infection in liverworts and angiosperms.
Project description:Background:The black pod disease affects cacao plantations worldwide; it is caused by the oomycete species of the genus Phytophthora. The resistance of cacao plants to the black pod is commonly evaluated by artificial inoculation of the pathogen and the monitoring of the disease symptoms. However, it is difficult to identify resistant plants because the commonly used methods for the inoculation of the pathogens produce inconsistent results. Therefore, this study aimed to develop an efficient and reliable method to evaluate the resistance of Theobroma cacao seedlings to the infection by Phytophthora palmivora. Results:Seedlings of different cacao genotypes were inoculated with P. palmivora under greenhouse conditions using the previously reported inoculation methods and a newly proposed method, the agar-water solution method. While none of the previously reported methods was effective, the agar-water solution method ensured a 100% seedling infection under greenhouse conditions. The proposed agar-water methodology is fast, simple and reproducible. Furthermore, the evaluation of this method in susceptible (CCN-51) and tolerant (SCA-6) T. cacao genotypes produced the expected contrasting results. Conclusions:The agar-water solution method presented in this study is an efficient alternative inoculation protocol for the identification of cacao genotypes that are resistant to black pod under greenhouse conditions.
Project description:The roots of most land plants are colonized by symbiotic arbuscular mycorrhiza (AM) fungi. To facilitate this symbiosis, plant genomes encode a set of genes required for microbial perception and accommodation. However, the extent to which infection by filamentous root pathogens also relies on some of these genes remains an open question. Here, we used genome-wide association mapping to identify genes contributing to colonization of Medicago truncatula roots by the pathogenic oomycete Phytophthora palmivora. Single-nucleotide polymorphism (SNP) markers most significantly associated with plant colonization response were identified upstream of RAD1, which encodes a GRAS transcription regulator first negatively implicated in root nodule symbiosis and recently identified as a positive regulator of AM symbiosis. RAD1 transcript levels are up-regulated both in response to AM fungus and, to a lower extent, in infected tissues by P. palmivora where its expression is restricted to root cortex cells proximal to pathogen hyphae. Reverse genetics showed that reduction of RAD1 transcript levels as well as a rad1 mutant are impaired in their full colonization by AM fungi as well as by P. palmivora. Thus, the importance of RAD1 extends beyond symbiotic interactions, suggesting a general involvement in M. truncatula microbe-induced root development and interactions with unrelated beneficial and detrimental filamentous microbes.
Project description:Rubber tree (Hevea brasiliensis Muell. Arg) is an important economic crop in Thailand. Leaf fall and black stripe diseases caused by the aggressive oomycete pathogen Phytophthora palmivora, cause deleterious damage on rubber tree growth leading to decrease of latex production. To gain insights into the molecular function of H. brasiliensis subtilisin-like serine proteases, the HbSPA, HbSPB, and HbSPC genes were transiently expressed in Nicotiana benthamiana via agroinfiltration. A functional protease encoded by HbSPA was successfully expressed in the apoplast of N. benthamiana leaves. Transient expression of HbSPA in N. benthamiana leaves enhanced resistance to P. palmivora, suggesting that HbSPA plays an important role in plant defense. P. palmivora Kazal-like extracellular protease inhibitor 10 (PpEPI10), an apoplastic effector, has been implicated in pathogenicity through the suppression of H. brasiliensis protease. Semi-quantitative RT-PCR revealed that the PpEPI10 gene was significantly up-regulated during colonization of rubber tree by P. palmivora. Concurrently, the HbSPA gene was highly expressed during infection. To investigate a possible interaction between HbSPA and PpEPI10, the recombinant PpEPI10 protein (rPpEPI10) was expressed in Escherichia coli and purified using affinity chromatography. In-gel zymogram and co-immunoprecipitation (co-IP) assays demonstrated that rPpEPI10 specifically inhibited and interacted with HbSPA. The targeting of HbSPA by PpEPI10 revealed a defense-counterdefense mechanism, which is mediated by plant protease and pathogen protease inhibitor, in H. brasiliensis-P. palmivora interactions.