Functional analysis of a Phytophthora host-translocated effector using the yeast model system

Background Phytophthora plant pathogens secrete effector proteins that are translocated into host plant cells during infection and collectively contribute to pathogenicity. A subset of these host-translocated effectors can be identified by the amino acid motif RXLR (arginine, any amino acid, leucine, arginine). Bioinformatics analysis has identified hundreds of putative RXLR effector genes in Phytophthora genomes, but the specific molecular function of most remains unknown. Methods Here we describe initial studies to investigate the use of Saccharomyces cerevisiae as a eukaryotic model to explore the function of Phytophthora RXLR effector proteins. Results and Conclusions Expression of individual RXLR effectors in yeast inhibited growth, consistent with perturbation of a highly conserved cellular process. Transcriptome analysis of yeast cells expressing the poorly characterized P. sojae RXLR effector Avh110 identified nearly a dozen yeast genes whose expression levels were altered greater than two-fold compared to control cells. All five of the most down-regulated yeast genes are normally induced under low phosphate conditions via the PHO4 transcription factor, indicating that PsAvh110 perturbs the yeast regulatory network essential for phosphate homeostasis and suggesting likely PsAvh110 targets during P. sojae infection of its soybean host.

Interaction of Phytophthora sojae Effector Avr1b With E3 Ubiquitin Ligase GmPUB1 Is Required for Recognition by Soybeans Carrying Phytophthora Resistance Rps1-b and Rps1-k Genes

Phytophthora sojae is an oomycete that causes stem and root rot disease in soybean. P. sojae delivers many RxLR effector proteins, including Avr1b, into host cells to promote infection. We show here that Avr1b interacts with the soybean U-box protein, GmPUB1-1, in yeast two-hybrid, pull down, and bimolecular fluorescence complementation (BIFC) assays. GmPUB1-1, and a homeologous copy GmPUB1-2, are induced by infection and encode 403 amino acid proteins with U-Box domains at their N-termini. Non-synonymous mutations in the Avr1b C-terminus that abolish suppression of cell death also abolished the interaction of Avr1b with GmPUB1-1, while deletion of the GmPUB1-1 C-terminus, but not the U box, abolished the interaction. BIFC experiments suggested that the GmPUB1-1-Avr1b complex is targeted to the nucleus. In vitro ubiquitination assays demonstrated that GmPUB1-1 possesses E3 ligase activity. Silencing of the GmPUB1 genes in soybean cotyledons resulted in loss of recognition of Avr1b by gene products encoded by Rps1-b and Rps1-k. The recognition of Avr1k (which did not interact with GmPUB1-1) by Rps1-k plants was not, however, affected following GmPUB1-1 silencing. Furthermore, over-expression of GmPUB1-1 in particle bombardment experiments triggered cell death suggesting that GmPUB1 may be a positive regulator of effector-triggered immunity. In a yeast two-hybrid system, GmPUB1-1 also interacted with a number of other RxLR effectors including Avr1d, while Avr1b and Avr1d interacted with a number of other infection-induced GmPUB proteins, suggesting that the pathogen uses a multiplex of interactions of RxLR effectors with GmPUB proteins to modulate host immunity.

Expression of several Phytophthora cinnamomi putative RxLRs provides evidence for virulence roles in avocado

Phytophthora cinnamomi is a plant pathogenic oomycete that causes Phytophthora root rot of avocado (PRR). Currently, there is a limited understanding of the molecular interactions underlying this disease. Other Phytophthora species employ an arsenal of effector proteins to manipulate host physiology, of which the RxLR effectors contribute to virulence by interfering with host immune responses. The aim of this study was to identify candidate RxLR effectors in P. cinnamomi that play a role in establishing PRR, and to infer possible functions for these effectors. We identified 61 candidate RxLR genes which were expressed during infection of a susceptible avocado rootstock. Several of these genes were present in multiple copies in the P. cinnamomi genome, suggesting that they may contribute to pathogen fitness. Phylogenetic analysis of the manually predicted RxLR protein sequences revealed 12 P. cinnamomi RxLRs that were related to characterised effectors in other Phytophthora spp., providing clues to their functions in planta. Expression profiles of nine more RxLRs point to possible virulence roles in avocado–highlighting a way forward for studies of this interaction. This study represents the first investigation of the expression of P. cinnamomi RxLR genes during the course of avocado infection, and puts forward a pipeline to pinpoint effector genes with potential as virulence determinants, providing a foundation for the future functional characterization of RxLRs that contribute to P. cinnamomi virulence in avocado.

Rpi-amr3 confers resistance to multiple Phytophthora species by recognizing a conserved RXLR effector

Diverse pathogens from the genus Phytophthora cause disease and reduce yields in many crop plants. Although many Resistance to Phytophthora infestans (Rpi) genes effective against potato late blight have been cloned, few have been cloned against other Phytophthora species. Most Rpi genes encode nucleotide-binding domain, leucine-rich repeat- containing (NLR) proteins, that recognize RXLR effectors. However, whether NLR proteins can recognize RXLR effectors from multiple different Phytophthora pathogens has rarely been investigated. Here, we report the effector AVRamr3 from P. infestans that is recognized by Rpi-amr3 from S. americanum. We show here that AVRamr3 is broadly conserved in many different Phytophthora species, and that recognition of AVRamr3 homologs enables resistance against multiple Phytophthora pathogens, including P. parasitica and P. palmivora. Our findings suggest a novel path to identifying R genes against important plant pathogens.

Genome Sequence Resource of Phytophthora colocasiae from China Using Nanopore Sequencing Technology

Phytophthora colocasiae is a destructive oomycete pathogen of taro (Colocasia esculenta), which causes taro leaf blight. To date, only one highly fragmented Illumina short-read-based genome assembly is available for this species. To address this problem, we sequenced strain Lyd2019 from China using Oxford Nanopore Technologies (ONT) long-read sequencing and Illumina short-read sequencing. We generated a 92.51-Mb genome assembly consisting of 105 contigs with an N50 of 1.70 Mb and a maximum length of 4.17 Mb. In the genome assembly, we identified 52.78% repeats and 18,322 protein-coding genes, of which 12,782 genes were annotated. We also identified 191 candidate RXLR effectors and 1 candidate CRN effectors. The updated near-chromosome genome assembly and annotation resources will provide a better understanding of the infection mechanisms of P. colocasiae.

Genome Sequence Resource of Phytophthora vignae, the causal agent of stem and root rot of cowpea

The causal agent of stem and root rot of cowpea, Phytophthora vignae, is a widely distributed species of Phytophthora genus. Here, we generate a high-quality complete genome assembly of P. vignae strain PSY2020 (89.39 Mb, N50: 2.99 Mb) from China using Oxford Nanopore Technologies (ONT) sequencing. The genome assembly completeness evaluated by BUSCO was 94.51% at eukaryote level. We identified 42.54% repeat sequences and a total of 20,536 protein-encoding genes, of which 15,184 genes could be annotated. And we also identified 924 candidate RXLR effectors in the genome assembly. The described genome sequence will provide a valuable resource for better understanding of pathogenicity mechanisms of P. vignae, and shedding light on uncovering phylogenetical classification of Phytophthora species.

Genome Sequence Data of Peronophythora litchii, an Oomycete Pathogen Causing Litchi Downy Blight

Peronophythora litchii is an oomycete pathogen that exclusively infects litchi, with infection stages affecting a broad range of tissues. In this study, we obtained a near chromosome-level genome assembly of P. litchii strain ZL2018 from China using Oxford Nanopore Technologies (ONT) long-read sequencing and Illumina short-read sequencing. The genome assembly was 64.15 Mb in size and consisted of 81 contigs with an N50 of 1.43 Mb and a maximum length of 4.74 Mb. Excluding 34.67% of repeat sequences, a total of 14,857 protein-coding genes were identified, among which 14,447 genes were annotated. We also predicted 306 candidate RXLR effectors in the assembly. The high-quality genome assembly and annotation resources reported in this study will provide new insight into the infection mechanisms of P. litchii.

First Insights into the Effect of Mycorrhizae on the Expression of Pathogen Effectors during the Infection of Grapevine with Plasmopara viticola

Grapevine (Vitis vinifera L.), widely used for berry and wine production, is highly susceptible to the pathogenic oomycete Plasmopara viticola, the etiological agent of grapevine downy mildew disease. The method commonly used to prevent and control P. viticola infection relies on multiple applications of chemical fungicides. However, with European Union goals to lower the usage of such chemicals in viticulture there is a need to develop new and more sustainable strategies. The use of beneficial microorganisms with biocontrol capabilities, such as the arbuscular mycorrhizal fungi (AMF), has been pointed out as a viable alternative. With this study, we intended to investigate the effect of AMF colonization on the expression of P. viticola effectors during infection of grapevine. Grapevine plants were inoculated with the AMF Rhizophagus irregularis and, after mycorrhizae development, plants were infected with P. viticola. The expression of P. viticola RxLR effectors was analyzed by real-time PCR (qPCR) during the first hours of interaction. Results show that pre-mycorrhizal inoculation of grapevine alters the expression of several P. viticola effectors; namely, PvRxLR28, which presented decreased expression in mycorrhizal plants at the two time points post-infection tested. These results suggest that the pre-inoculation of grapevine with AMF could interfere with the pathogen’s ability to infect grapevine by modulation of pathogenicity effectors expression, supporting the hypothesis that AMF can be used to increase plant resistance to pathogens and promote more sustainable agriculture practices, particularly in viticulture.

Allelic variants of the NLR protein Rpi-chc1 differentially recognise members of the Phytophthora infestans PexRD12/31 effector superfamily through the leucine-rich repeat domain

SummaryPhytophthora infestans is a pathogenic oomycete that causes the infamous potato late blight disease. Resistance (R) genes from diverse Solanum species encode intracellular receptors that recognize P. infestans RXLR effector proteins and provide effective defence responses. To deploy these R genes in a durable fashion in agriculture, we need to understand the mechanism of effector recognition and the way the pathogen evades recognition.We cloned sixteen allelic variants of the Rpi-chc1 gene from Solanum chacoense and other Solanum species, and identified the cognate P. infestans RXLR effectors. These tools were used to study receptor-ligand interactions and co-evolution.Functional and non-functional alleles of Rpi-chc1 encode Coiled-Coil-Nucleotide Binding-Leucine-Rich-Repeat (CNL) proteins. Rpi-chc1.1 recognised multiple PexRD12 (AVRchc1.1) proteins while Rpi-chc1.2 recognised multiple PexRD31 (AVRchc1.2) proteins, both from the PexRD12/31 superfamily. Domain swaps between Rpi-chc1.1 and Rpi-chc1.2 revealed that overlapping subdomains in the LRR were responsible for the difference in effector recognition.This study showed that Rpi-chc1.1 and Rpi-chc1.2, evolved to recognize distinct members of the same PexRD12/31 effector family via the LRR domain. The biased distribution of polymorphisms suggests that exchange of LRRs during host-pathogen co-evolution can lead to novel recognition specificities. These insights will help future strategies to breed for durable resistant varieties.