Search Results (11)

Phytophthora cinnamomi is a major plant pathogen that affects economically important crops and natural ecosystems, posing a threat to global biodiversity. While gene editing has emerged as a powerful tool for functional genomics in various Phytophthora species, its application in P. cinnamomi remains underexplored. To address this gap, our study investigated the challenges of implementing CRISPR/Cas9-mediated gene editing in P. cinnamomi, with the insights gained applicable to other gene editing platforms. We designed guide RNAs (gRNAs) targeting β-cinnamomin, a highly basic elicitin expressed by the pathogen during early infection stages, known for its role in sterol recruitment. Using an “all-in-one” plasmid containing the gRNA, Cas9, and an antibiotic resistance gene as a selectable marker, we transformed P. cinnamomi protoplasts via PEG/CaCl₂-mediated methods. The successful integration of the nptII gene, which confers geneticin (G418) resistance, was confirmed in heterokaryotic regenerants. To isolate pure mutants and eliminate wild-type dominance, we derived homokaryotic colonies from nptII-positive transformants. Mutation screening was performed using mismatch detection assays, T7 endonuclease 1 (T7E1), and restriction fragment length polymorphism (RFLP), followed by Sanger sequencing. Despite the integration of the nptII gene, the β-cinnamomin sequence in the transformants remained identical to the wild-type sequence, indicating challenges in detecting and achieving targeted gene disruption. This study identifies critical steps for optimising mutagenesis in P. cinnamomi, highlighting the importance of thorough preliminary screening, effective separation of heterokaryotic populations, and the isolation of homokaryotic colonies. Full article

Bud Rot, caused by Phytophthora palmivora, is considered one of the main diseases affecting African oil palm (Elaeis guineensis). In this study, we investigated the in vitro molecular dynamics of the pathogen–host interaction by analyzing gene expression profiles from oil palm genotypes that were either susceptible or resistant to the disease. We observed distinct interactions of P. palmivora with resistant and susceptible oil palms through co-expression network analysis. When interacting with susceptible genotypes, P. palmivora exhibited upregulation of carbohydrate and sulfate transport genes. These genes demonstrated co-expression with apoplastic and cytoplasmic effectors, including cell wall degrading enzymes, elicitins, and RxLR motif effectors. The pathogen manipulated susceptible oil palm materials, exacerbating the response and compromising the phenylpropanoid pathway, ultimately leading to susceptibility. In contrast, resistant materials exhibited control over their response through putative Heat Shock Proteins (HSP) that maintained homeostasis between primary metabolism and biotic defense. Co-expressed genes related to flavonoids, WRKY transcripts, lectin-type receptors, and LRR receptors may play important roles in pathogen control. Overall, the study provides new knowledge of the molecular mechanisms underlying the interaction between E. guineensis and P. palmivora, which can contribute to controlling Bud Rot in oil palms and gives new insights into the interactions of P. palmivora with their hosts. Full article

Phytophthora infestans, an Oomycete pathogen, has a devastating impact on potato agriculture, leading to the extensive use of chemical fungicides to prevent its outbreaks. Spraying double-stranded RNAs to suppress specific genes of the pathogen via the RNA interference (RNAi) pathway may provide an environmentally friendly alternative to chemicals. However, this novel approach will require various target genes and application strategies to be tested. Using the L4440 backbone, we have designed two plasmids to express dsRNA targeting inf1 and inf4 genes of P. infestans that are known to contribute to the disease development at different stages. The dsRNA produced by the bacteria was tested on potato explants and demonstrated a statistically significant reduction in lesions five days after inoculation compared to water treatment. The study results allow us to consider our approach to be promising for potato late blight control. Full article

In interactions between pathogens and plants, pathogens secrete many molecules that facilitate plant infection, and some of these compounds are recognized by plant pattern recognition receptors (PRRs), which induce immune responses. Molecules in both pathogens and plants that trigger immune responses in plants are termed elicitors. On the basis of their chemical content, elicitors can be classified into carbohydrates, lipopeptides, proteinaceous compounds and other types. Although many studies have focused on the involvement of elicitors in plants, especially on pathophysiological changes induced by elicitors in plants and the mechanisms mediating these changes, there is a lack of up-to-date reviews on the characteristics and functions of proteinaceous elicitors. In this mini-review, we provide an overview of the up-to-date knowledge on several important families of pathogenic proteinaceous elicitors (i.e., harpins, necrosis- and ethylene-inducing peptide 1 (nep1)-like proteins (NLPs) and elicitins), focusing mainly on their structures, characteristics and effects on plants, specifically on their roles in plant immune responses. A solid understanding of elicitors may be helpful to decrease the use of agrochemicals in agriculture and gardening, generate more resistant germplasms and increase crop yields. Full article

Phytophthora cactorum is an important oomycetous plant pathogen with numerous host plant species, including garden strawberry (Fragaria × ananassa) and silver birch (Betula pendula). P. cactorum also hosts mycoviruses, but their phenotypic effects on the host oomycete have not been studied earlier. In the present study, we tested polyethylene glycol (PEG)-induced water stress for virus curing and created an isogenic virus-free isolate for testing viral effects in pair with the original isolate. Phytophthora cactorum bunya-like viruses 1 and 2 (PcBV1 & 2) significantly reduced hyphal growth of the P. cactorum host isolate, as well as sporangia production and size. Transcriptomic and proteomic analyses revealed an increase in the production of elicitins due to bunyavirus infection. However, the presence of bunyaviruses did not seem to alter the pathogenicity of P. cactorum. Virus transmission through anastomosis was unsuccessful in vitro. Full article

In contrast to most pathogenic oomycetes, which infect plants, Pythium insidiosum infects both humans and animals, causing a difficult-to-treat condition called pythiosis. Most patients undergo surgical removal of an affected organ, and advanced cases could be fetal. As a successful human/animal pathogen, P. insidiosum must tolerate body temperature and develop some strategies to survive and cause pathology within hosts. One of the general pathogen strategies is virulence factor secretion. Here, we used proteogenomic analysis to profile and validate the secretome of P. insidiosum, in which its genome contains 14,962 predicted proteins. Shotgun LC–MS/MS analysis of P. insidiosum proteins prepared from liquid cultures incubated at 25 and 37 °C mapped 2980 genome-predicted proteins, 9.4% of which had a predicted signal peptide. P. insidiosum might employ an alternative secretory pathway, as 90.6% of the validated secretory/extracellular proteins lacked the signal peptide. A comparison of 20 oomycete genomes showed 69 P. insidiosum–specific secretory/extracellular proteins, and these may be responsible for the host-specific infection. The differential expression analysis revealed 14 markedly upregulated proteins (particularly cyclophilin and elicitin) at body temperature which could contribute to pathogen fitness and thermotolerance. Our search through a microbial virulence database matched 518 secretory/extracellular proteins, such as urease and chaperones (including heat shock proteins), that might play roles in P. insidiosum virulence. In conclusion, the identification of the secretome promoted a better understanding of P. insidiosum biology and pathogenesis. Cyclophilin, elicitin, chaperone, and urease are top-listed secreted/extracellular proteins with putative pathogenicity properties. Such advances could lead to developing measures for the efficient detection and treatment of pythiosis. Full article

Pathogens and plants are in a constant battle with one another, the result of which is either the restriction of pathogen growth via constitutive or induced plant defense responses or the pathogen colonization of plant cells and tissues that cause disease. Elicitins are a group of highly conserved proteins produced by certain oomycete species, and their sterol binding ability is recognized as an important feature in sterol–auxotrophic oomycetes. Elicitins also orchestrate other aspects of the interactions of oomycetes with their plant hosts. The function of elicitins as avirulence or virulence factors is controversial and is dependent on the host species, and despite several decades of research, the function of these proteins remains elusive. We summarize here our current understanding of elicitins as either defense-promoting or defense-suppressing agents and propose that more recent approaches such as the use of ‘omics’ and gene editing can be used to unravel the role of elicitins in host–pathogen interactions. A better understanding of the role of elicitins is required and deciphering their role in host–pathogen interactions will expand the strategies that can be adopted to improve disease resistance and reduce crop losses. Full article

Plants are constantly challenged by various environmental stressors ranging from abiotic—sunlight, elevated temperatures, drought, and nutrient deficits, to biotic factors—microbial pathogens and insect pests. These not only affect the quality of harvest but also the yield, leading to substantial annual crop losses, worldwide. Although plants have a multi-layered immune system, phytopathogens such as species of the oomycete genus Phytophthora, can employ elaborate mechanisms to breach this defense. For the last two decades, researchers have focused on the co-evolution between Phytophthora and interacting hosts to decouple the mechanisms governing their molecular associations. This has provided a comprehensive understanding of the pathobiology of plants affected by oomycetes. Ultimately, this is important for the development of strategies to sustainably improve agricultural production. Therefore, this paper discusses the present-day state of knowledge of the strategic mode of operation employed by species of Phytophthora for successful infection. Specifically, we consider motility, attachment, and host cell wall degradation used by these pathogenic species to obtain nutrients from their host. Also discussed is an array of effector types from apoplastic (hydrolytic proteins, protease inhibitors, elicitins) to cytoplastic (RxLRs, named after Arginine-any amino acid-Leucine-Arginine consensus sequence and CRNs, for CRinkling and Necrosis), which upon liberation can subvert the immune response and promote diseases in plants. Full article

Phytophthora cinnamomi is a devastating pathogen causing root and crown rot and dieback diseases of nearly 5000 plant species. Accurate and rapid detection of P. cinnamomi plays a fundamental role within the current disease prevention and management programs. In this study, a novel effector gene PHYCI_587572 was found as unique to P. cinnamomi based on a comparative genomic analysis of 12 Phytophthora species. Its avirulence homolog protein 87 (Avh87) is characterized by the Arg-Xaa-Leu-Arg (RxLR) motif. Avh87 suppressed the pro-apoptotic protein BAX- and elicitin protein INF1-mediated cell death of Nicotiana benthamiana. Furthermore, a recombinase polymerase amplification-lateral flow dipstick detection assay targeting this P. cinnamomi-specific biomarker was developed. While successfully detected 19 P. cinnamomi isolates of a global distribution, this assay lacked detection of 37 other oomycete and fungal species, including P. parvispora, a sister taxon of P. cinnamomi. In addition, it detected P. cinnamomi from artificially inoculated leaves of Cedrus deodara. Moreover, the RPA-LFD assay was found to be more sensitive than a conventional PCR assay, by detecting as low as 2 pg of genomic DNA in a 50-µL reaction. It detected P. cinnamomi in 13 infested soil samples, while the detection rate was 46.2% using PCR. Results in this study indicated that PHYCI_587572 is a unique biomarker for detecting P. cinnamomi. Although PHYCI_587572 was identified as an effector gene based on the RxLR motif of Avh87 and the avirulence activity on Nicotiana, its exact genetic background and biological function on the natural hosts of P. cinnamomi warrant further investigations. Full article

‘Candidatus Liberibacter asiaticus’ (CLas) is one of the causal agents of citrus Huanglongbing (HLB), a bacterial disease of citrus trees that greatly reduces fruit yield and quality. CLas strains produce an array of currently uncharacterized Sec-dependent secretory proteins. In this study, the conserved chromosomally encoded protein CLIBASIA_03875 was identified as a novel Sec-dependent secreted protein. We show that CLIBASIA_03875 contains a putative Sec- secretion signal peptide (SP), a 29 amino acid residue located at the N-terminus, with a mature protein (m3875) of 22 amino acids found to localize in multiple subcellular components of the leaf epidermal cells of Nicotiana benthamiana. When overexpressed via a Potato virus X (PVX)-based expression vector in N. benthamiana, m3875 suppressed programmed cell death (PCD) and the H₂O₂ accumulation triggered by the pro-apoptotic mouse protein BAX and the Phytophthora infestans elicitin INF1. Overexpression also resulted in a phenotype of dwarfing, leaf deformation and mosaics, suggesting that m3875 has roles in plant immune response, growth, and development. Substitution mutagenesis of the charged amino acid (D7, R9, R11, and K22) with alanine within m3875 did not recover the phenotypes for PCD and normal growth. In addition, the transiently overexpressed m3875 regulated the transcriptional levels of N. benthamiana orthologs of CNGCs (cyclic nucleotide-gated channels), BI-1 (Bax-inhibitor 1), and WRKY33 that are involved in plant defense mechanisms. To our knowledge, m3875 is the first PCD suppressor identified from CLas. Studying the function of this protein provides insight as to how CLas attenuates the host immune responses to proliferate and cause Huanglongbing disease in citrus plants. Full article

The induction of plant immunity by Pathogen Associated Molecular Patterns (PAMPs) constitutes a powerful strategy for crop protection. PAMPs indeed induce general defense responses in plants and thus increase plant resistance to pathogens. Phytophthora infestans culture filtrates (CCFs) are known to induce defense responses and decrease the severity of soft rot due to Pectobacterium atrosepticum in potato tubers. The aim of this study was to identify and characterize the active compounds from P. infestans filtrate. The filtrate was fractionated by gel filtration, and the protection effects against P. atrosepticum and the ability to induce PAL activity were tested for each fraction. The fraction active in protection (F1) also induced PAL activity, as did the whole filtrate. Three elicitins (INF1, INF4 and INF5) were identified in F1b, subfraction of F1, by MALDI-TOF-MS and MS/MS analyses. However, deproteinized F1b still showed biological activity against the bacterium, revealing the presence of an additional active compound. GC-MS analyses of the deproteinized fraction highlighted the presence of a galactan-based complex polysaccharide. These experiments demonstrate that the biological activity of the CCF against P. atrosepticum results from a combined action of three elicitins and a complex polysaccharide, probably through the activation of general defense responses. Full article