An RxLR effector from phytophthora infestans prevents re-localisation of two plant NAC transcription factors from the endoplasmic reticulum to the nucleus

The plant immune system is activated following the perception of exposed, essential and invariant microbial molecules that are recognised as non-self. A major component of plant immunity is the transcriptional induction of genes involved in a wide array of defence responses. In turn, adapted pathogens deliver effector proteins that act either inside or outside plant cells to manipulate host processes, often through their direct action on plant protein targets. To date, few effectors have been shown to directly manipulate transcriptional regulators of plant defence. Moreover, little is known generally about the modes of action of effectors from filamentous (fungal and oomycete) plant pathogens. We describe an effector, called Pi03192, from the late blight pathogen Phytophthora infestans, which interacts with a pair of host transcription factors at the endoplasmic reticulum (ER) inside plant cells. We show that these transcription factors are released from the ER to enter the nucleus, following pathogen perception, and are important in restricting disease. Pi03192 prevents the plant transcription factors from accumulating in the host nucleus, revealing a novel means of enhancing host susceptibility

A molecular insight into algal-oomycete warfare : cDNA analysis of Ectocarpus siliculosus infected with the basal oomycete Eurychasma dicksonii

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Short interspersed nuclear element (SINE) sequences in the genome of the human pathogenic fungus Aspergillus fumigatus Af293.

Copyright: © 2016 The Authors. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Citation: Kanhayuwa L, Coutts RHA (2016) Short Interspersed Nuclear Element (SINE) Sequences in the Genome of the Human Pathogenic Fungus Aspergillus fumigatus Af293. PLoS ONE 11(10): e0163215. https://doi.org/10.1371/journal.pone.0163215.Novel families of short interspersed nuclear element (SINE) sequences in the human pathogenic fungus Aspergillus fumigatus, clinical isolate Af293, were identified and categorised into tRNA-related and 5S rRNA-related SINEs. Eight predicted tRNA-related SINE families originating from different tRNAs, and nominated as AfuSINE2 sequences, contained target site duplications of short direct repeat sequences (4-14 bp) flanking the elements, an extended tRNA-unrelated region and typical features of RNA polymerase III promoter sequences. The elements ranged in size from 140-493 bp and were present in low copy number in the genome and five out of eight were actively transcribed. One putative tRNAArg-derived sequence, AfuSINE2-1a possessed a unique feature of repeated trinucleotide ACT residues at its 3'-terminus. This element was similar in sequence to the I-4_AO element found in A. oryzae and an I-1_AF long nuclear interspersed element-like sequence identified in A. fumigatus Af293. Families of 5S rRNA-related SINE sequences, nominated as AfuSINE3, were also identified and their 5'-5S rRNA-related regions show 50-65% and 60-75% similarity to respectively A. fumigatus 5S rRNAs and SINE3-1_AO found in A. oryzae. A. fumigatus Af293 contains five copies of AfuSINE3 sequences ranging in size from 259-343 bp and two out of five AfuSINE3 sequences were actively transcribed. Investigations on AfuSINE distribution in the fungal genome revealed that the elements are enriched in pericentromeric and subtelomeric regions and inserted within gene-rich regions. We also demonstrated that some, but not all, AfuSINE sequences are targeted by host RNA silencing mechanisms. Finally, we demonstrated that infection of the fungus with mycoviruses had no apparent effects on SINE activity.Peer reviewedFinal Published versio

Sequence Variants of the Phytophthora sojae RXLR Effector Avr3a/5 Are Differentially Recognized by Rps3a and Rps5 in Soybean

The perception of Phytophthora sojae avirulence (Avr) gene products by corresponding soybean resistance (Rps) gene products causes effector triggered immunity. Past studies have shown that the Avr3a and Avr5 genes of P. sojae are genetically linked, and the Avr3a gene encoding a secreted RXLR effector protein was recently identified. We now provide evidence that Avr3a and Avr5 are allelic. Genetic mapping data from F2 progeny indicates that Avr3a and Avr5 co-segregate, and haplotype analysis of P. sojae strain collections reveal sequence and transcriptional polymorphisms that are consistent with a single genetic locus encoding Avr3a/5. Transformation of P. sojae and transient expression in soybean were performed to test how Avr3a/5 alleles interact with soybean Rps3a and Rps5. Over-expression of Avr3a/5 in a P. sojae strain that is normally virulent on Rps3a and Rps5 results in avirulence to Rps3a and Rps5; whereas silencing of Avr3a/5 causes gain of virulence in a P. sojae strain that is normally avirulent on Rps3a and Rps5 soybean lines. Transient expression and co-bombardment with a reporter gene confirms that Avr3a/5 triggers cell death in Rps5 soybean leaves in an appropriate allele-specific manner. Sequence analysis of the Avr3a/5 gene identifies crucial residues in the effector domain that distinguish recognition by Rps3a and Rps5

Identification and rapid mapping of a gene conferring broad-spectrum late blight resistance in the diploid potato species <i>Solanum verrucosum</i> through DNA capture technologies

Key message: A broad-spectrum late blight disease-resistance gene from Solanum verrucosum has been mapped to potato chromosome 9. The gene is distinct from previously identified-resistance genes. Abstract: We have identified and characterised a broad-spectrum resistance to Phytophthora infestans from the wild Mexican species Solanum verrucosum. Diagnostic resistance gene enrichment (dRenSeq) revealed that the resistance is not conferred by previously identified nucleotide-binding, leucine-rich repeat genes. Utilising the sequenced potato genome as a reference, two complementary enrichment strategies that target resistance genes (RenSeq) and single/low-copy number genes (Generic-mapping enrichment Sequencing; GenSeq), respectively, were deployed for the rapid, SNP-based mapping of the resistance through bulked-segregant analysis. Both approaches independently positioned the resistance, referred to as Rpi-ver1, to the distal end of potato chromosome 9. Stringent post-enrichment read filtering identified a total of 64 informative SNPs that corresponded to the expected ratio for significant polymorphisms in the parents as well as the bulks. Of these, 61 SNPs are located on potato chromosome 9 and reside within 27 individual genes, which in the sequenced potato clone DM locate to positions 45.9 to 60.9 Mb. RenSeq- and GenSeq-derived SNPs within the target region were converted into allele-specific PCR-based KASP markers and further defined the position of the resistance to a 4.3 Mb interval at the bottom end of chromosome 9 between positions 52.62–56.98 Mb.</p

Functionally Redundant RXLR Effectors from <em>Phytophthora infestans</em> Act at Different Steps to Suppress Early flg22-Triggered Immunity

Genome sequences of several economically important phytopathogenic oomycetes have revealed the presence of large families of so-called RXLR effectors. Functional screens have identified RXLR effector repertoires that either compromise or induce plant defense responses. However, limited information is available about the molecular mechanisms underlying the modes of action of these effectors in planta. The perception of highly conserved pathogen- or microbe-associated molecular patterns (PAMPs/MAMPs), such as flg22, triggers converging signaling pathways recruiting MAP kinase cascades and inducing transcriptional re-programming, yielding a generic anti-microbial response. We used a highly synchronizable, pathogen-free protoplast-based assay to identify a set of RXLR effectors from Phytophthora infestans (PiRXLRs), the causal agent of potato and tomato light blight that manipulate early stages of flg22-triggered signaling. Of thirty-three tested PiRXLR effector candidates, eight, called Suppressor of early Flg22-induced Immune response (SFI), significantly suppressed flg22-dependent activation of a reporter gene under control of a typical MAMP-inducible promoter (pFRK1-Luc) in tomato protoplasts. We extended our analysis to Arabidopsis thaliana, a non-host plant species of P. infestans. From the aforementioned eight SFI effectors, three appeared to share similar functions in both Arabidopsis and tomato by suppressing transcriptional activation of flg22-induced marker genes downstream of post-translational MAP kinase activation. A further three effectors interfere with MAMP signaling at, or upstream of, the MAP kinase cascade in tomato, but not in Arabidopsis. Transient expression of the SFI effectors in Nicotiana benthamiana enhances susceptibility to P. infestans and, for the most potent effector, SFI1, nuclear localization is required for both suppression of MAMP signaling and virulence function. The present study provides a framework to decipher the molecular mechanisms underlying the manipulation of host MAMP-triggered immunity (MTI) by P. infestans and to understand the basis of host versus non-host resistance in plants towards P. infestans

Cell entry of a host targeting protein of oomycetes requires gp96

This work is supported by the [European Community’s] Seventh Framework Programme [FP7/2007–2013] under grant agreement no. [238550] (L.L., J.D.-U., C.J.S., P.v.W.); BBSRC [BBE007120/1, BB/J018333/1 and BB/G012075/1] (F.T., I.d.B., C.J.S., S.W., P.v.W.); Newton Global Partnership Award [BB/N005058/1] (F.T., P.v.W.), the University of Aberdeen (A.D.T., T.R., C.J.S., P.v.W.) and Deutsche Forschungsgemeinschaft [CRC1093] (P.B., T.S.). We would like to acknowledge the Ministry of Higher Education Malaysia for funding INA. We would like to thank Brian Haas for his bioinformatics support. We would like to acknowledge Neil Gow and Johannes van den Boom for critical reading of the manuscript. We would like to acknowledge Svetlana Rezinciuc for technical help with pH-studies.Peer reviewedPublisher PD

Bridging the Gulf: Phytophthora and Downy Mildews Are Connected by Rare Grass Parasites

Downy mildews and root and foliar rots caused by Phytophthora are among the most destructive plant pathogens and therefore have attracted considerable attention during the past two decades. Although it has been realized that a close phylogenetic relationship exists, so far sharp distinction has been made between the obligate biotrophic downy mildews and the hemibiotrophic Phytophthora. In the study presented here, it is shown that a continuum of character states from hemibiotrophic Phytophthora species to obligate biotrophic downy mildews is present. Intermediate character states between downy mildews and Phytophthora species exist in several rare parasites of grasses, which are not embedded within the major clades of the downy mildews but are placed sister to these, with unresolved affinities to both these clades and to Phytophthora. They still have retained traits hitherto thought to be exclusive for Phytophthora. A careful review of previous research is presented and it is highlighted that uniquely for downy mildews, Poakatesthia may form an intracellular mycelium, growing through several host cells. In addition, scanning electron microscopy reveals that sporangiophore growth is not determinate in Viennotia and that outgrowth from sporangiophores is very similar to Phytophthora infestans. It is concluded that the sharp morphological distinction between downy mildews and Phytophthora species (that are often placed in separate families and even different orders), is rather artificial, since all features thought to be exclusive to Phytophthora or the downy mildews are united in the rare grass-parasitizing down mildew genera Viennotia and Poakatesthia and the enigmatic genus Sclerophthora. Therefore, several paradigms regarding the distinction between Phytophthora and the downy mildews need to be reconsidered

Competition between Phytophthora infestans Effectors Leads to Increased Aggressiveness on Plants Containing Broad-Spectrum Late Blight Resistance

BACKGROUND: The destructive plant disease potato late blight is caused by the oomycete pathogen Phytophthora infestans (Mont.) de Bary. This disease has remained particularly problematic despite intensive breeding efforts to integrate resistance into cultivated potato, largely because of the pathogen's ability to quickly evolve to overcome major resistance genes. The RB gene, identified in the wild potato species S. bulbocastanum, encodes a protein that confers broad-spectrum resistance to most P. infestans isolates through its recognition of highly conserved members of the corresponding pathogen effector family IPI-O. IpiO is a multigene family of effectors and while the majority of IPI-O proteins are recognized by RB to elicit host resistance, some variants exist that are able to elude detection (e.g. IPI-O4). METHODS AND FINDINGS: In the present study, analysis of ipiO variants among 40 different P. infestans isolates collected from Guatemala, Thailand, and the United States revealed a high degree of complexity within this gene family. Isolate aggressiveness was correlated with increased ipiO diversity and especially the presence of the ipiO4 variant. Furthermore, isolates expressing IPI-O4 overcame RB-mediated resistance in transgenic potato plants even when the resistance-eliciting IPI-O1 variant was present. In support of this finding, we observed that expression of IPI-O4 via Agrobacterium blocked recognition of IPI-O1, leading to inactivation of RB-mediated programmed cell death in Nicotiana benthamiana. CONCLUSIONS: In this study we definitively demonstrate and provide the first evidence that P. infestans can defeat an R protein through inhibition of recognition of the corresponding effector protein