RNA silencing proteins and small RNAs in oomycete plant pathogens and biocontrol agents

IntroductionOomycetes cause several damaging diseases of plants and animals, and some species also act as biocontrol agents on insects, fungi, and other oomycetes. RNA silencing is increasingly being shown to play a role in the pathogenicity of Phytophthora species, either through trans-boundary movement of small RNAs (sRNAs) or through expression regulation of infection promoting effectors. MethodsTo gain a wider understanding of RNA silencing in oomycete species with more diverse hosts, we mined genome assemblies for Dicer-like (DCL), Argonaute (AGO), and RNA dependent RNA polymerase (RDRP) proteins from Phytophthora plurivora, Ph. cactorum, Ph. colocasiae, Pythium oligandrum, Py. periplocum, and Lagenidium giganteum. Moreover, we sequenced small RNAs from the mycelium stage in each of these species. Results and discussionEach of the species possessed a single DCL protein, but they differed in the number and sequence of AGOs and RDRPs. SRNAs of 21nt, 25nt, and 26nt were prevalent in all oomycetes analyzed, but the relative abundance and 5' base preference of these classes differed markedly between genera. Most sRNAs mapped to transposons and other repeats, signifying that the major role for RNA silencing in oomycetes is to limit the expansion of these elements. We also found that sRNAs may act to regulate the expression of duplicated genes. Other sRNAs mapped to several gene families, and this number was higher in Pythium spp., suggesting a role of RNA silencing in regulating gene expression. Genes for most effector classes were the source of sRNAs of variable size, but some gene families showed a preference for specific classes of sRNAs, such as 25/26 nt sRNAs targeting RxLR effector genes in Phytophthora species. Novel miRNA-like RNAs (milRNAs) were discovered in all species, and two were predicted to target transcripts for RxLR effectors in Ph. plurivora and Ph. cactorum, indicating a putative role in regulating infection. Moreover, milRNAs from the biocontrol Pythium species had matches in the predicted transcriptome of Phytophthora infestans and Botrytis cinerea, and L. giganteum milRNAs matched candidate genes in the mosquito Aedes aegypti. This suggests that trans-boundary RNA silencing may have a role in the biocontrol action of these oomycetes

RNA silencing proteins and small RNAs in oomycete plant pathogens and biocontrol agents

Introduction: Oomycetes cause several damaging diseases of plants and animals, and some species also act as biocontrol agents on insects, fungi, and other oomycetes. RNA silencing is increasingly being shown to play a role in the pathogenicity of Phytophthora species, either through trans-boundary movement of small RNAs (sRNAs) or through expression regulation of infection promoting effectors.Methods: To gain a wider understanding of RNA silencing in oomycete species with more diverse hosts, we mined genome assemblies for Dicer-like (DCL), Argonaute (AGO), and RNA dependent RNA polymerase (RDRP) proteins from Phytophthora plurivora, Ph. cactorum, Ph. colocasiae, Pythium oligandrum, Py. periplocum, and Lagenidium giganteum. Moreover, we sequenced small RNAs from the mycelium stage in each of these species.Results and discussion: Each of the species possessed a single DCL protein, but they differed in the number and sequence of AGOs and RDRPs. SRNAs of 21nt, 25nt, and 26nt were prevalent in all oomycetes analyzed, but the relative abundance and 5’ base preference of these classes differed markedly between genera. Most sRNAs mapped to transposons and other repeats, signifying that the major role for RNA silencing in oomycetes is to limit the expansion of these elements. We also found that sRNAs may act to regulate the expression of duplicated genes. Other sRNAs mapped to several gene families, and this number was higher in Pythium spp., suggesting a role of RNA silencing in regulating gene expression. Genes for most effector classes were the source of sRNAs of variable size, but some gene families showed a preference for specific classes of sRNAs, such as 25/26 nt sRNAs targeting RxLR effector genes in Phytophthora species. Novel miRNA-like RNAs (milRNAs) were discovered in all species, and two were predicted to target transcripts for RxLR effectors in Ph. plurivora and Ph. cactorum, indicating a putative role in regulating infection. Moreover, milRNAs from the biocontrol Pythium species had matches in the predicted transcriptome of Phytophthora infestans and Botrytis cinerea, and L. giganteum milRNAs matched candidate genes in the mosquito Aedes aegypti. This suggests that trans-boundary RNA silencing may have a role in the biocontrol action of these oomycetes.</p

Oomycete interactions with plants: infection strategies and resistance principles.

The Oomycota include many economically significant microbial pathogens of crop species. Understanding the mechanisms by which oomycetes infect plants and identifying methods to provide durable resistance are major research goals. Over the last few years, many elicitors that trigger plant immunity have been identified, as well as host genes that mediate susceptibility to oomycete pathogens. The mechanisms behind these processes have subsequently been investigated and many new discoveries made, marking a period of exciting research in the oomycete pathology field. This review provides an introduction to our current knowledge of the pathogenic mechanisms used by oomycetes, including elicitors and effectors, plus an overview of the major principles of host resistance: the established R gene hypothesis and the more recently defined susceptibility (S) gene model. Future directions for development of oomycete-resistant plants are discussed, along with ways that recent discoveries in the field of oomycete-plant interactions are generating novel means of studying how pathogen and symbiont colonizations overlap.The authors acknowledge funding from the Gatsby Charitable Foundation (GAT3273/GLD). SF and SS acknowledge funding by the Royal Society. SF would also like to acknowledge personal funding from The Morley Agricultural Foundation and The Felix Cobbold Trust.This is the accepted manuscript. The final version is available at http://mmbr.asm.org/content/79/3/263.abstract

Small RNAs in Phytophthora infestans and cross-talk with potato

Small RNAs (sRNAs) are small non-coding RNAs usually ranging in size 20-30 nt. They are playing important roles in plant-pathogen interactions. This thesis aimed at studying the sRNA populations in potato and Phytophthora infestans and their role in the potato-P. infestans interaction. An attempt was also made to implement such knowledge to improve resistance in potato against P. infestans. P. infestans is an oomycete that causes severe damage to potato and tomato, and is well known for its ability to evolve rapidly to overcome resistance. It possesses active RNA silencing pathways and sRNAs are playing important roles to control the large numbers of transposable elements (TE) present in its genome and effector genes. Through deep sequencing of sRNAs from two isolates of P. infestans differing in pathogenicity, three clear size classes (21, 25/26 and 32 nt) of sRNAs were identified. RxLR and Crinkler (CRN) effector gene-derived sRNAs were present in both isolates, but exhibited marked differences in abundance. Some effector genes, such as PiAvr3a and PiAvrblb2, to which sRNAs were found, also exhibited differences in transcript accumulation between the two isolates. Majority of sRNAs also mapped to TEs. An additional group of sRNAs, the tRNA-derived RNA fragments (tRFs) ranging in size from 19-40 nt was identified in P. infestansm as well. Some tRFs accumulated differentially during infection. A host-induced gene-silencing (HIGS) approach was proven to be successful in the potato-P. infestans pathosystem. Four different endogenous genes in P. infestans were targeted in my study by HIGS constructs and choice of target gene was shown crucial for a successful outcome. HIGS has the potential to be adopted in new resistance breeding to improve resistance in potato against P. infestans. One late blight resistant potato cultivar was sequenced upon infection with compatible isolate of P. infestans. In order to decipher the molecular events underlying its resistance breakdown, analysis of transcript change and the possible role of sRNAs on transcript regulation during infection are ongoing. In infected potato, resistance genes are targeted by its own miRNAs leading to suppression. Additional components are most likely involved in this process and near future analysis will help to enhance our understanding of how P. infestans uses sRNAs to evade plant immune responses

Effector vector design in the Phytophthora infestans-potato pathosystem

The oomycete pathogen Phytophthora infestans is the causal agent of the devastating plant disease late blight on potato. Diverse type of transposons and many gene families are present in the genome which encodes the effector proteins involved in causing the pathogenicity. This plant pathogen is predicted to secret hundreds of effector proteins inside the host plant cells to promote infection. These proteins are sensed by the plant immune system in order to prevent pathogen growth. The effector proteins are divided into two main types, cytoplasmic effectors and apoplastic effectors based on their translocated status in the plant cell. In this study, the effectorencoding genes Avr3a, Epi1, Epi10, Inf1 and CRN8 were selected to monitor the potential in planta function of the effectors and to develop a stable transformation procedure for reporter gene constructs with effector gene promoters. The putative promoter sequences were derived from the 5´ regions of the oomycete genes. Primers were designed to amplify the promoter regions and the amplification was confirmed by gel electrophoresis. The reporter gene GFP (encoding green fluorescent protein) was chosen for analysis of their promoter activities and to facilitate studies on spatial and dynamic alteration of gene expression. Cloning was performed using the vector pTOR-eGFP containing a ham34 promoter and a GFP gene. The ham34 promoter was removed and the effector promoters were inserted in its place. A stable transformation procedure was examined using three vectors for the GFP-constructs and the five effector gene promoters. Transformants were obtained at similar frequencies with each combination of effector promoter and GFP; which were confirmed by gel electrophoresis. Subsequently Agrobacterium tumefaciens (C58) mediated transformation was tried for an Avr3a promoter construct. The construct was ligated into the binary vector, but the transformation of Agrobacterium was not successful

The Phytophthora infestans avirulence gene PiaAvr4 and its potato counterpart R4

The potato late blight disease that is caused by the oomycete pathogen Phytophthora infestans is a major threat for potato crops worldwide. In recent years research on oomycete plant pathogens was boosted by the availability of novel genomic tools and resources for several oomycete genera, such as Phytophthora, Hyaloperonospora, Pythium and Aphanomyces. This has led to the identification of genes involved in diverse biological processes such as sporulation, mating, signaling and pathogenesis. One of the approaches that breeders use to obtain late blight resistant potato cultivars is the introgression of resistance traits from wild Solanum species into the cultivated potato Solanum tuberosum. The pathogen, however, is able to circumvent this resistance; it is often lost shortly after introduction of new cultivars. To better understand the mechanisms underlying this loss of resistance it is of utmost importance to gain insight into the characteristics of the cognate avirulence (Avr) genes of the pathogen. According to the gene-for-gene model Avr genes encode effectors that trigger resistance responses mediated by resistance (R) genes. This thesis first describes the identification of a P. infestans Avr gene, in particular the elicitor activity of the encoded effector protein, the domain structure of the effector and its putative sub-cellular localization. In the second part the recognition specificity of the corresponding R gene and the identification of a marker linked to this R gene are described. Chapter 1 summarizes the advances in oomycete genomics in recent years and the tremendous progress that has been made in gene discovery in oomycete plant pathogens. It describes the different oomycete species that have been studied in more detail and assesses which species are suitable model species for research on oomycete-plant interactions. The identification of the P. infestans avirulence gene PiAvr4 is presented in Chapter 2. PiAvr4, which encodes an RXLR-dEER effector protein, was isolated by positional cloning. AFLP markers were used for landing on BACs and cDNA-AFLP markers pinpointed the gene of interest. Transformation of race 4 strains with PiAvr4 resulted in transformants that are avirulent on the R4 differential of the Mastenbroek differential set (clone Ma-R4). Moreover, in planta expression of PiAvr4 resulted in a necrotic response on clone Ma-R4 but not on plants lacking R4 such as Bintje. All together this proves that PiAvr4 is the avirulence gene that corresponds to the R gene present in clone Ma-R4. In many identified avirulence proteins one or a few amino acid changes in the protein abolish avirulence function. In case of PiAvr4, race 4 strains have frame shift mutations in the open reading frame, resulting in a truncated protein that is not functional as avirulence factor. Effectors within the RXLR-dEER family are rapidly evolving. The selective pressure is targeted predominantly on the C-terminal region of these proteins. Despite this selective pressure the majority of these proteins carry motifs that can be distinguished using Hidden Markov Models searches. They are named W, Y and L motifs after the conserved tryptophan (W), tyrosine (Y) and leucine (L) residues, respectively. As described in Chapter 3 PiAvr4 carries three W motifs and a single Y motif. The motifs together with their flanking regions were tested for activity on Ma-R4 plants. Agroinfection of constructs carrying the W2 motif in combination with either the W1 or W3 motif resulted in a necrotic response. Moreover, we showed that the PiAvr4 homolog PmirAvh4, isolated from Phytophthora mirabilis was also able to elicit a necrotic response on the Ma-R4 potato clone. For several Phytophthora RXLR-dEER effectors it was demonstrated that these proteins are targeted into the host cell and that the RXLR-dEER motif is required for translocation. In Chapter 4 we investigated whether PiAvr4 and IPI-O, like other RXLR-dEER effectors, are also targeted into the host cell. A race 4 P. infestans isolate was transformed with constructs encoding either PiAvr4 or IPI-O fused to a monomeric red fluorescent protein (mRFP) at the C-terminus. Fluorescence microscopy of these transformants showed no specific mRFP fluorescence in free living, non-sporulating mycelium. However, in germinating cysts, the tips of germ tubes and appressoria showed mRFP fluorescence, and during infection of etiolated potato plantlets localized fluorescence was visible at the haustorial neck. Haustoria are highly specialized infection and feeding structures that are in close contact with the plant cell and have a putative role in delivering effector proteins into the host cell. In order to monitor the development of the infection a novel experimental set-up was developed. In this method etiolated in vitro grown potato plantlets are inoculated with P. infestans, which has the advantage that there is no autofluorescence of chlorophyll that masks the mRFP fluorescence and thus disturbs the microscopic analysis in green plant tissues. The lack of chlorophyll does not seem to interfere with infection; zoospores are capable to encyst and to germinate, and the etiolated tissues are readily colonized by P. infestans. The recognition specificity of R4 potato differentials is described in Chapter 5. Initially two different potato clones were developed as R4 differentials; The Mastenbroek differential set, developed in the Netherlands, contains the clone Cebeco44-31-5 (designated as Ma-R4) and the Black differential set, developed in Scotland, contains clone 1563 c (14) (designated as Bl-R4). Virulence assays using several wild type P. infestans strains revealed that the Bl-R4 clone is susceptible to all isolates that are avirulent on clone Ma-R4. Only one single isolate was found to be avirulent on clone Bl-R4, but virulent on Ma-R4. Moreover, in transient expression assays with binary PVX constructs carrying PiAvr4, the Ma-R4 clone but not the Bl-R4 clone responded with an HR. Similar to the R3 locus two different recognition specificities seem to exist for R4. The R3a and R3b genes are located on one locus but whether this is the case for the two R4 genes (named R4Ma and R4Bl, respectively) remains to be determined. Resistance to P. infestans strains carrying PiAvr4 segregates in an 1:1 ratio in two independent potato F1 populations suggesting that R4Ma resistance is determined by a single dominant locus. More in depth studies on the recognition of PiAvr4 by its cognate R protein are hampered by the fact that the resistance gene R4Ma has not yet been identified. In Chapter 6 nucleotide binding site (NBS) profiling was used to generate R4Ma-associated markers. NBS profiling is a biased approach based on PCR amplification of conserved NBS motifs in R genes and R gene homologs. In a bulked segregant analysis, DNA of resistant and susceptible F1 progeny was pooled and used as template for NBS profiling. Several candidate markers were found but eventually one amplified fragment was found to co-segregate with resistance mediated by R4Ma. DNA sequencing of this fragment revealed high similarity to BAC sequences that are mapped to potato chromosome 12. Moreover, the R4Ma marker is homologous to members of the Rx/Gpa2 gene family. Chapter 7 focuses on the secreted effectors of plant pathogenic oomycetes, with special attention to RXLR-dEER effectors, and the role of these proteins in pathogenesis. The RXLR-dEER effector family is rapidly evolving and comprises all secreted oomycete avirulence proteins that are identified up till now. There is now ample evidence that oomycetes utilize the RXLR-dEER domain to deposit effectors inside host cells. Furthermore, this chapter discusses the experimental results described in this thesis in the light of present knowledge on gene-for-gene interactions, effector recognition and late blight resistance. <br/

Genetic modification to improve disease resistance in crops

Plant pathogens are a significant challenge in agriculture despite our best efforts to combat them. One of the most effective and sustainable ways to manage plant pathogens is to use genetic modification (GM) and genome editing, expanding the breeder's toolkit. For use in the field, these solutions must be efficacious, with no negative effect on plant agronomy, and deployed thoughtfully. They must also not introduce a potential allergen or toxin. Expensive regulation of biotech crops is prohibitive for local solutions. With 11-30% average global yield losses and greater local impacts, tackling plant pathogens is an ethical imperative. We need to increase world food production by at least 60% using the same amount of land, by 2050. The time to act is now and we cannot afford to ignore the new solutions that GM provides to manage plant pathogens. This article is protected by copyright. All rights reserved

Isolation and characterization of potato homologues of Arabidopsis thaliana genes operating in defense signal transduction

An increasing number of pathogen-defense related genes are being identified and characterized in Arabidopsis thaliana. So far, it is not known whether and which structural and functional homologues of these Arabidopsis genes have any role in natural variation of resistance to pathogens in crops. Using sequence database mining and PCR-based approaches, potato (Solanum tuberosum L.) gene fragments with high sequence similarity to 16 Arabidopsis defense signal transduction genes were obtained, sequenced and genetically positioned on potato molecular maps. Of 16 novel loci, five were positional candidates for known potato pathogen resistance QTL. One of the candidate loci, StAOS2 co-localizing with QTL for resistance to P. infestans and E. carotovora on linkage group XI, was further characterized in more detail. StAOS2 encodes a gene for allene oxide synthase, a cytochrome P450-enzyme, acting upstream in the jasmonic acid biosynthesis pathway. A metabolic block at the level of AOS completely abolishes JA production, which affects plant development (e.g. sterile pollen production) and various abiotic and biotic stress responses (e.g. P. infestans resistance in tomato, E. carotovora resistance in Arabidopsis). The chloroplastic localization of StAOS2-GFP was confirmed by confocal microscopy and functionality of the potato protein was proven by complementation of the male-sterile Arabidopsis aos mutant. StAOS2-RNAi transgenic lines in potato were generated in order to test role of StAOS2 in P. infestans resistance. The measurements of endogenous OPDA and JA in the silenced lines after wounding treatment revealed drastic decrease in the levels of above mentioned compounds (up to 25 folds less than in wild type plants). In addition, natural variation of StAOS2 locus was characterized. Sequencing of the locus across 38 potato chromosomes revealed high polymorphism. Thirteen distinct alleles were found, and four of them showed highly significant (P=0.000, R2=14%) linkage to P. infestans and E. carotovora QTL. Five alleles of StAOS2 were cloned. Sequence analyses revealed a substantial polymorphism on amino acid level, including non-conservative substitutions and an insertion/deletion within the cytochrome P450 domain. Currently, an ongoing quantitative complementation of the Ataos mutant with the five different StAOS2 alleles fused to the native AtAOS promoter, followed by OPDA and JA levels measurements in the transgenic lines, will possibly provide direct evidence for StAOS2 being the first plant resistance QTL identified

The molecular dialog between oomycete effectors and their plant and animal hosts

Funding Information: This work was supported by European Union's HORIZON 2020 Research programme under the Grant Agreement no. 766048 “PROTECTA”, University of Aberdeen (PvW), Wageningen University and Research (VGAAV) , the BBSRC [ BB/P020224/1 , BB/M026566/1 (MS, PvW)], Newton Fund GRP Aquaculture [ BB/N005058/1 (PvW)], and the Peruvian Council for science, technology and technological innovation (CONCYTEC) FONDECYT contract 129–2017 .Peer reviewedPublisher PD