CRN Effectors Research Articles

Comparative Genomic and Secretome Analysis of Phytophthora capsici Strains: Exploring Pathogenicity and Evolutionary Dynamics

Phytophthora capsici is a destructive oomycete pathogen that poses a significant threat to global agriculture by infecting a wide range of economically important crops in the Solanaceae and Cucurbitaceae families. In Canada, the pathogen has been responsible for substantial losses in greenhouse and field-grown crops. Despite extensive worldwide research on P. capsici, little is known about the effector content and pathogenicity of the Canadian isolates. In this study, we sequenced and analyzed the genomes of two Canadian P. capsici strains, namely 55330 and 55898, and conducted a comparative secretome analysis with globally referenced strains LT1534 and LT263. The Canadian strains displayed smaller genomes at 57.3 Mb and 60.2 Mb compared to LT263 at 76 Mb, yet retained diverse effector repertoires, including RxLR and CRN effectors, and exhibited robust pathogenic potential. Our analysis revealed that while the Canadian strains have fewer unique effector clusters compared to LT263, they possess comparable CAZyme profiles, emphasizing their capacity to degrade plant cell walls and promote infection. The differences in effector content likely reflect host adaptation, as P. capsici infects a variety of plant species. This study provides valuable insights into the genetic features of Canadian P. capsici isolates and offers a foundation for future efforts in developing targeted disease-management strategies.

Identification of Phytophthora cinnamomi CRN effectors and their roles in manipulating cell death during Persea americana infection

The oomycete Phytophthora cinnamomi is a devastating plant pathogen with a notably broad host range. It is the causal agent of Phytophthora root rot (PRR), arguably the most economically important yield-limiting disease in Persea americana (avocado). Despite this, our understanding of the mechanisms P. cinnamomi employs to infect and successfully colonize avocado remains limited, particularly regarding the pathogen’s ability to maintain its biotrophic and necrotrophic lifestyles during infection. The pathogen utilises a large repertoire of effector proteins which function in facilitating and establishing disease in susceptible host plants. Crinkling and necrosis effectors (CRN/Crinklers) are suspected to manipulate cell death to aid in maintenance of the pathogens biotrophic and necrotrophic lifestyles during different stages of infection. The current study identified 25 P. cinnamomi CRN effectors from the GKB4 genome using an HMM profile and assigned putative function to them as either cell death inducers or suppressors. Function was assigned to 10 PcinCRNs by analysing their RNA-seq expression profiles, relatedness to other functionally characterised Phytophthora CRNs and tertiary protein predictions. The full-length coding sequences for these PcinCRNs were confirmed by Sanger sequencing, six of which were found to have two divergent alleles. The presence of alleles indicates that the proteins encoded may perform contradicting functions in cell death manipulation, or function in different host plant species. Overall, this study provides a foundation for future research on P. cinnamomi infection and cell death manipulation mechanisms.

Genome Sequence Resource for Phytopythium helicoides, the Causal Pathogen of Crown and Root Rot of Photinia × fraseri.

The oomycete Phytopythium helicoides is a globally distributed plant pathogen, and causes crown blight, root rot and seedling damping in many agricultural plants. P. helicoides PF-he2 was isolated from infected Photinia × fraseri Dress in China. Here, a high-quality genome of PF-he2 was sequenced by a combination of PacBio and Illumina sequencing strategies. The genome is 49.09 Mb in length with 105 contigs. The N50 contig length is 860 kb, and BUSCO completeness is 94%. Gene prediction resulted in 16,807 protein-coding genes, and 1,663 secreted proteins were identified. In addition, we identified a series of proteins involved in pathogenicity, including 30 CRN effectors, 26 YxSL[RK] effectors, 30 NLP proteins, and 49 elicitin-like proteins. This genome provides a valuable resource for better understanding the genetic diversity and molecular basis of pathogenesis of P. helicoides, which will facilitate the development of effective control strategies.

Chromosome-level assembly of the Phytophthora agathidicida genome reveals adaptation in effector gene families.

Phytophthora species are notorious plant pathogens, with some causing devastating tree diseases that threaten the survival of their host species. One such example is Phytophthora agathidicida, the causal agent of kauri dieback - a root and trunk rot disease that kills the ancient, iconic and culturally significant tree species, Agathis australis (New Zealand kauri). A deeper understanding of how Phytophthora pathogens infect their hosts and cause disease is critical for the development of effective treatments. Such an understanding can be gained by interrogating pathogen genomes for effector genes, which are involved in virulence or pathogenicity. Although genome sequencing has become more affordable, the complete assembly of Phytophthora genomes has been problematic, particularly for those with a high abundance of repetitive sequences. Therefore, effector genes located in repetitive regions could be truncated or missed in a fragmented genome assembly. Using a combination of long-read PacBio sequences, chromatin conformation capture (Hi-C) and Illumina short reads, we assembled the P. agathidicida genome into ten complete chromosomes, with a genome size of 57 Mb including 34% repeats. This is the first Phytophthora genome assembled to chromosome level and it reveals a high level of syntenic conservation with the complete genome of Peronospora effusa, the only other completely assembled genome sequence of an oomycete. All P. agathidicida chromosomes have clearly defined centromeres and contain candidate effector genes such as RXLRs and CRNs, but in different proportions, reflecting the presence of gene family clusters. Candidate effector genes are predominantly found in gene-poor, repeat-rich regions of the genome, and in some cases showed a high degree of duplication. Analysis of candidate RXLR effector genes that occur in multicopy gene families indicated half of them were not expressed in planta. Candidate CRN effector gene families showed evidence of transposon-mediated recombination leading to new combinations of protein domains, both within and between chromosomes. Further analysis of this complete genome assembly will help inform new methods of disease control against P. agathidicida and other Phytophthora species, ultimately helping decipher how Phytophthora pathogens have evolved to shape their effector repertoires and how they might adapt in the future.

Karyotype variation, spontaneous genome rearrangements affecting chemical insensitivity, and expression level polymorphisms in the plant pathogen Phytophthora infestans revealed using its first chromosome-scale assembly.

Natural isolates of the potato and tomato pathogen Phytophthora infestans exhibit substantial variation in virulence, chemical sensitivity, ploidy, and other traits. A chromosome-scale assembly was developed to expand genomic resources for this oomyceteous microbe, and used to explore the basis of variation. Using PacBio and Illumina data, a long-range linking library, and an optical map, an assembly was created and coalesced into 15 pseudochromosomes spanning 219 Mb using SNP-based genetic linkage data. De novo gene prediction combined with transcript evidence identified 19,981 protein-coding genes, plus about eight thousand tRNA genes. The chromosomes were comprised of a mosaic of gene-rich and gene-sparse regions plus very long centromeres. Genes exhibited a biased distribution across chromosomes, especially members of families encoding RXLR and CRN effectors which clustered on certain chromosomes. Strikingly, half of F1 progeny of diploid parents were polyploid or aneuploid. Substantial expression level polymorphisms between strains were identified, much of which could be attributed to differences in chromosome dosage, transposable element insertions, and adjacency to repetitive DNA. QTL analysis identified a locus on the right arm of chromosome 3 governing sensitivity to the crop protection chemical metalaxyl. Strains heterozygous for resistance often experienced megabase-sized deletions of that part of the chromosome when cultured on metalaxyl, increasing resistance due to loss of the sensitive allele. This study sheds light on diverse phenomena affecting variation in P. infestans and relatives, helps explain the prevalence of polyploidy in natural populations, and provides a new foundation for biologic and genetic investigations.

Transcriptome analysis of Phytophthora litchii reveals pathogenicity arsenals and confirms taxonomic status.

Litchi downy blight, caused by Peronophythora litchii, is one of the major diseases of litchi and has caused severe economic losses. P. litchii has the unique ability to produce downy mildew like sporangiophores under artificial culture. The pathogen had been placed in a new family Peronophytophthoraceae by some authors. In this study, the whole transcriptome of P. litchii from mycelia, sporangia, and zoospores was sequenced for the first time. A set of 23637 transcripts with an average length of 1284 bp was assembled. Using six open reading frame (ORF) predictors, 19267 representative ORFs were identified and were annotated by searching against several public databases. There were 4666 conserved gene families and various sets of lineage-specific genes among P. litchii and other four closely related oomycetes. In silico analyses revealed 490 pathogen-related proteins including 128 RXLR and 22 CRN effector candidates. Based on the phylogenetic analysis of 164 single copy orthologs from 22 species, it is validated that P. litchii is in the genus Phytophthora. Our work provides valuable data to elucidate the pathogenicity basis and ascertain the taxonomic status of P. litchii.

The 25-26 nt Small RNAs in Phytophthora parasitica Are Associated with Efficient Silencing of Homologous Endogenous Genes.

Small RNAs (sRNAs) are important non-coding RNA regulators, playing key roles in developmental regulation, transposon suppression, environmental response, host–pathogen interaction and other diverse biological processes. However, their roles in oomycetes are poorly understood. Here, we performed sRNA sequencing and RNA sequencing of Phytophthora parasitica at stages of vegetative growth and infection of Arabidopsis roots to examine diversity and function of sRNAs in P. parasitica, a model hemibiotrophic oomycete plant pathogen. Our results indicate that there are two distinct types of sRNA-generating loci in P. parasitica genome, giving rise to clusters of 25–26 nt and 21 nt sRNAs, respectively, with no significant strand-biases. The 25–26 nt sRNA loci lie predominantly in gene-sparse and repeat-rich regions, and overlap with over 7000 endogenous gene loci. These overlapped genes are typically P. parasitica species-specific, with no homologies to the sister species P. infestans. They include approximately 40% RXLR effector genes, 50% CRN effector genes and some elicitor genes. The transcripts of most of these genes could not be detected at both the vegetative mycelium and infection stages as revealed by RNA sequencing, indicating that the 25–26 nt sRNAs are associated with efficient silencing of these genes. The 21 nt sRNA loci typically overlap with the exon regions of highly expressed genes, suggesting that the biogenesis of the 21 nt sRNAs may be dependent on the level of gene transcription and that these sRNAs do not mediate efficient silencing of homologous genes. Analyses of the published P. infestans sRNA and mRNA sequencing data consistently show that the 25–26 nt sRNAs, but not the 21 nt sRNAs, may mediate efficient gene silencing in Phytophthora.

Genome sequence of Plasmopara viticola and insight into the pathogenic mechanism

Plasmopara viticola causes downy mildew disease of grapevine which is one of the most devastating diseases of viticulture worldwide. Here we report a 101.3 Mb whole genome sequence of P. viticola isolate ‘JL-7-2’ obtained by a combination of Illumina and PacBio sequencing technologies. The P. viticola genome contains 17,014 putative protein-coding genes and has ~26% repetitive sequences. A total of 1,301 putative secreted proteins, including 100 putative RXLR effectors and 90 CRN effectors were identified in this genome. In the secretome, 261 potential pathogenicity genes and 95 carbohydrate-active enzymes were predicted. Transcriptional analysis revealed that most of the RXLR effectors, pathogenicity genes and carbohydrate-active enzymes were significantly up-regulated during infection. Comparative genomic analysis revealed that P. viticola evolved independently from the Arabidopsis downy mildew pathogen Hyaloperonospora arabidopsidis. The availability of the P. viticola genome provides a valuable resource not only for comparative genomic analysis and evolutionary studies among oomycetes, but also enhance our knowledge on the mechanism of interactions between this biotrophic pathogen and its host.

RXLR and CRN Effectors from the Sunflower Downy Mildew Pathogen Plasmopara halstedii Induce Hypersensitive-Like Responses in Resistant Sunflower Lines.

Plasmopara halstedii is an obligate biotrophic oomycete causing downy mildew disease on sunflower, Helianthus annuus, an economically important oil crop. Severe symptoms of the disease (e.g., plant dwarfism, leaf bleaching, sporulation and production of infertile flower) strongly impair seed yield. Pl resistance genes conferring resistance to specific P. halstedii pathotypes were located on sunflower genetic map but yet not cloned. They are present in cultivated lines to protect them against downy mildew disease. Among the 16 different P. halstedii pathotypes recorded in France, pathotype 710 is frequently found, and therefore continuously controlled in sunflower by different Pl genes. High-throughput sequencing of cDNA from P. halstedii led us to identify potential effectors with the characteristic RXLR or CRN motifs described in other oomycetes. Expression of six P. halstedii putative effectors, five RXLR and one CRN, was analyzed by qRT-PCR in pathogen spores and in the pathogen infecting sunflower leaves and selected for functional analyses. We developed a new method for transient expression in sunflower plant leaves and showed for the first time subcellular localization of P. halstedii effectors fused to a fluorescent protein in sunflower leaf cells. Overexpression of the CRN and of 3 RXLR effectors induced hypersensitive-like cell death reactions in some sunflower near-isogenic lines resistant to pathotype 710 and not in susceptible corresponding lines, suggesting they could be involved in Pl loci-mediated resistances.

A Phytophthora sojae effector PsCRN63 forms homo-/hetero-dimers to suppress plant immunity via an inverted association manner.

Oomycete pathogens produce a large number of effectors to promote infection. Their mode of action are largely unknown. Here we show that a Phytophthora sojae effector, PsCRN63, suppresses flg22-induced expression of FRK1 gene, a molecular marker in pathogen-associated molecular patterns (PAMP)-triggered immunity (PTI). However, PsCRN63 does not suppress upstream signaling events including flg22-induced MAPK activation and BIK1 phosphorylation, indicating that it acts downstream of MAPK cascades. The PsCRN63-transgenic Arabidopsis plants showed increased susceptibility to bacterial pathogen Pseudomonas syringae pathovar tomato (Pst) DC3000 and oomycete pathogen Phytophthora capsici. The callose deposition were suppressed in PsCRN63-transgenic plants compared with the wild-type control plants. Genes involved in PTI were also down-regulated in PsCRN63-transgenic plants. Interestingly, we found that PsCRN63 forms an dimer that is mediated by inter-molecular interactions between N-terminal and C-terminal domains in an inverted association manner. Furthermore, the N-terminal and C-terminal domains required for the dimerization are widely conserved among CRN effectors, suggesting that homo-/hetero-dimerization of Phytophthora CRN effectors is required to exert biological functions. Indeed, the dimerization was required for PTI suppression and cell death-induction activities of PsCRN63.

Dual RNA-Sequencing of Eucalyptus nitens during Phytophthora cinnamomi Challenge Reveals Pathogen and Host Factors Influencing Compatibility.

Damage caused by Phytophthora cinnamomi Rands remains an important concern on forest tree species. The pathogen causes root and collar rot, stem cankers, and dieback of various economically important Eucalyptus spp. In South Africa, susceptible cold tolerant Eucalyptus plantations have been affected by various Phytophthora spp. with P. cinnamomi considered one of the most virulent. The molecular basis of this compatible interaction is poorly understood. In this study, susceptible Eucalyptus nitens plants were stem inoculated with P. cinnamomi and tissue was harvested five days post inoculation. Dual RNA-sequencing, a technique which allows the concurrent detection of both pathogen and host transcripts during infection, was performed. Approximately 1% of the reads mapped to the draft genome of P. cinnamomi while 78% of the reads mapped to the Eucalyptus grandis genome. The highest expressed P. cinnamomi gene in planta was a putative crinkler effector (CRN1). Phylogenetic analysis indicated the high similarity of this P. cinnamomi CRN1 to that of Phytophthora infestans. Some CRN effectors are known to target host nuclei to suppress defense. In the host, over 1400 genes were significantly differentially expressed in comparison to mock inoculated trees, including suites of pathogenesis related (PR) genes. In particular, a PR-9 peroxidase gene with a high similarity to a Carica papaya PR-9 ortholog previously shown to be suppressed upon infection by Phytophthora palmivora was down-regulated two-fold. This PR-9 gene may represent a cross-species effector target during P. cinnamomi infection. This study identified pathogenicity factors, potential manipulation targets, and attempted host defense mechanisms activated by E. nitens that contributed to the susceptible outcome of the interaction.

Effector Polymorphisms of the Sunflower Downy Mildew Pathogen Plasmopara halstedii and Their Use to Identify Pathotypes from Field Isolates.

The obligate biotroph oomycete Plasmopara halstedii causes downy mildew on sunflower crop, Helianthus annuus. The breakdown of several Pl resistance genes used in sunflower hybrids over the last 25 years came along with the appearance of new Pl. halstedii isolates showing modified virulence profiles. In oomycetes, two classes of effector proteins, key players of pathogen virulence, are translocated into the host: RXLR and CRN effectors. We identified 54 putative CRN or RXLR effector genes from transcriptomic data and analyzed their genetic diversity in seven Pl. halstedii pathotypes representative of the species variability. Pl. halstedii effector genes were on average more polymorphic at both the nucleic and protein levels than random non-effector genes, suggesting a potential adaptive dynamics of pathogen virulence over the last 25 years. Twenty-two KASP (Competitive Allele Specific PCR) markers designed on polymorphic effector genes were genotyped on 35 isolates belonging to 14 Pl. halstedii pathotypes. Polymorphism analysis based on eight KASP markers aims at proposing a determination key suitable to classify the eight multi-isolate pathotypes into six groups. This is the first report of a molecular marker set able to discriminate Pl. halstedii pathotypes based on the polymorphism of pathogenicity effectors. Compared to phenotypic tests handling living spores used until now to discriminate Pl. halstedii pathotypes, this set of molecular markers constitutes a first step in faster pathotype diagnosis of Pl. halstedii isolates. Hence, emerging sunflower downy mildew isolates could be more rapidly characterized and thus, assessment of plant resistance breakdown under field conditions should be improved.

An Oomycete CRN Effector Reprograms Expression of Plant HSP Genes by Targeting their Promoters.

Oomycete pathogens produce a large number of CRN effectors to manipulate plant immune responses and promote infection. However, their functional mechanisms are largely unknown. Here, we identified a Phytophthora sojae CRN effector PsCRN108 which contains a putative DNA-binding helix-hairpin-helix (HhH) motif and acts in the plant cell nucleus. Silencing of the PsCRN108 gene reduced P. sojae virulence to soybean, while expression of the gene in Nicotiana benthamiana and Arabidopsis thaliana enhanced plant susceptibility to P. capsici. Moreover, PsCRN108 could inhibit expression of HSP genes in A. thaliana, N. benthamiana and soybean. Both the HhH motif and nuclear localization signal of this effector were required for its contribution to virulence and its suppression of HSP gene expression. Furthermore, we found that PsCRN108 targeted HSP promoters in an HSE- and HhH motif-dependent manner. PsCRN108 could inhibit the association of the HSE with the plant heat shock transcription factor AtHsfA1a, which initializes HSP gene expression in response to stress. Therefore, our data support a role for PsCRN108 as a nucleomodulin in down-regulating the expression of plant defense-related genes by directly targeting specific plant promoters.

Comparative analysis of expressed CRN and RXLR effectors from two Plasmopara species causing grapevine and sunflower downy mildew

Plasmopara halstedii and Plasmopara viticola are the causative agents of downy mildew of two economically important crops, sunflower and grapevine, respectively. These phylogenetically related oomycetes are obligate biotrophs belonging to Peronosporales but have different pathological profiles. Despite the economic importance of diseases caused by both Plasmopara, genomic resources are very limited and their effector repertoire is unknown. In this study parallel transcriptome sequencing of P. halstedii and P. viticola was performed, and a Plasmopara species cDNA database (PlasmoparaSp) released containing 46 000 clusters. In oomycetes, two classes of effectors are translocated into the host cytoplasm, the RXLRs and the CRNs. The PlasmoparaSp database was screened in order to identify the repertoire of expressed effectors used by both Plasmopara species; about 50 putative RXLR and 60 CRN were identified for each. These effectors were compared within both species and with seven publicly available oomycete species representative of Peronosporales and Albuginales. Sequence analyses revealed the presence of 55 RXLR families, 12 of them shared by both Plasmopara, and 19 showing amino acid conservation with predicted peptides from at least one oomycete species. Analyses of Plasmopara spp. CRN C‐terminal variable regions revealed sequence conservation inside Plasmopara spp. and across oomycetes, with the exception of Hyaloperonospora arabidopsidis. Finally, the analyses confirmed the presence of eight CRN C‐terminal domains described in Phytophthora infestans and identified CRN effectors showing similarity to serine proteases. Species‐specific effectors were identified that may be involved in host specificity, as well as effectors conserved among oomycetes that might play important roles in the pathogen biology.

Profiling the Secretome and Extracellular Proteome of the Potato Late Blight Pathogen Phytophthora infestans

Oomycetes are filamentous organisms that cause notorious diseases, several of which have a high economic impact. Well known is Phytophthora infestans, the causal agent of potato late blight. Previously, in silico analyses of the genome and transcriptome of P. infestans resulted in the annotation of a large number of genes encoding proteins with an N-terminal signal peptide. This set is collectively referred to as the secretome and comprises proteins involved in, for example, cell wall growth and modification, proteolytic processes, and the promotion of successful invasion of plant cells. So far, proteomic profiling in oomycetes was primarily focused on subcellular, intracellular or cell wall fractions; the extracellular proteome has not been studied systematically. Here we present the first comprehensive characterization of the in vivo secretome and extracellular proteome of P. infestans. We have used mass spectrometry to analyze P. infestans proteins present in seven different growth media with mycelial cultures and this resulted in the consistent identification of over two hundred proteins. Gene ontology classification pinpointed proteins involved in cell wall modifications, pathogenesis, defense responses, and proteolytic processes. Moreover, we found members of the RXLR and CRN effector families as well as several proteins lacking an obvious signal peptide. The latter were confirmed to be bona fide extracellular proteins and this suggests that, similar to other organisms, oomycetes exploit non-conventional secretion mechanisms to transfer certain proteins to the extracellular environment.

Quantitative Label-Free Phosphoproteomics of Six Different Life Stages of the Late Blight Pathogen Phytophthora infestans Reveals Abundant Phosphorylation of Members of the CRN Effector Family

The oomycete Phytophthora infestans is the causal agent of late blight in potato and tomato. Since the underlying processes that govern pathogenicity and development in P. infestans are largely unknown, we have performed a large-scale phosphoproteomics study of six different P. infestans life stages. We have obtained quantitative data for 2922 phosphopeptides and compared their abundance. Life-stage-specific phosphopeptides include ATP-binding cassette transporters and a kinase that only occurs in appressoria. In an extended data set, we identified 2179 phosphorylation sites and deduced 22 phosphomotifs. Several of the phosphomotifs matched consensus sequences of kinases that occur in P. infestans but not Arabidopsis. In addition, we detected tyrosine phosphopeptides that are potential targets of kinases resembling mammalian tyrosine kinases. Among the phosphorylated proteins are members of the RXLR and Crinkler effector families. The latter are phosphorylated in several life stages and at multiple positions, in sites that are conserved between different members of the Crinkler family. This indicates that proteins in the Crinkler family have functions beyond their putative role as (necrosis-inducing) effectors. This phosphoproteomics data will be instrumental for studies on oomycetes and host-oomycete interactions. The data sets have been deposited to ProteomeXchange (identifier PXD000433).

Correction: Identification and Characterisation CRN Effectors in Phytophthora capsici Shows Modularity and Functional Diversity

Correction: Identification and Characterisation CRN Effectors in Phytophthora capsici Shows Modularity and Functional Diversity

Characterization of cell death inducing Phytophthora capsici CRN effectors suggests diverse activities in the host nucleus

Plant-Microbe interactions are complex associations that feature recognition of Pathogen Associated Molecular Patterns by the plant immune system and dampening of subsequent responses by pathogen encoded secreted effectors. With large effector repertoires now identified in a range of sequenced microbial genomes, much attention centers on understanding their roles in immunity or disease. These studies not only allow identification of pathogen virulence factors and strategies, they also provide an important molecular toolset suited for studying immunity in plants. The Phytophthora intracellular effector repertoire encodes a large class of proteins that translocate into host cells and exclusively target the host nucleus. Recent functional studies have implicated the CRN protein family as an important class of diverse effectors that target distinct subnuclear compartments and modify host cell signaling. Here, we characterized three necrosis inducing CRNs and show that there are differences in the levels of cell death. We show that only expression of CRN20_624 has an additive effect on PAMP induced cell death but not AVR3a induced ETI. Given their distinctive phenotypes, we assessed localization of each CRN with a set of nuclear markers and found clear differences in CRN subnuclear distribution patterns. These assays also revealed that expression of CRN83_152 leads to a distinct change in nuclear chromatin organization, suggesting a distinct series of events that leads to cell death upon over-expression. Taken together, our results suggest diverse functions carried by CRN C-termini, which can be exploited to identify novel processes that take place in the host nucleus and are required for immunity or susceptibility.

Nuclear components and dynamics during plant innate immunity

In plants, efficient immune responses against microbial infection depend on the ability to rapidly couple pathogen recognition to downstream signaling responses. In this context, plant immunity requires highly dynamic responses that involve multiple organelles during the recognition and signaling events associated with defense. Nuclear dynamics play a critical role in plant immunity based on the growing number of reports revealing that nuclear localization of pathogen effectors, plant disease resistance proteins, and key plant components, including transcription factors and regulators, are essential for immunity. This Research Topic provides an overview of the current knowledge about the importance of nuclear components—both from the “microbial side” and from the “plant side”—and nuclear dynamics during the establishment of plant immune responses. Mutations in plant cellular factors involved in the transport of macromolecules through the nuclear envelope compromise plant resistance signaling, underlining the importance of nucleocytoplasmic trafficking during plant innate immunity. The Mini-Review article by Gaouar and Germain (2013) describes the importance of nuclear mRNA export during plant immune responses whereas Wirthmueller et al. (2013) discuss importin-α-mediated nuclear translocation and how microbial effectors may compete with host cargo proteins for nuclear uptake. Following their delivery into plant cells, a significant number of effector proteins from different pathogenic microorganisms, including viruses, oomycetes, fungi, nematodes, and bacteria, are targeted to the nucleus by co-opting the host nuclear import machinery. This suggests that effectors may manipulate host transcription or directly target essential host nuclear components for the benefit of the pathogen. Indeed, pathogen-induced transcriptional regulation in host cells plays a crucial role in the establishment of plant defense and associated plant cell death responses. Several articles in this Research Topic highlight these ideas. Quentin et al. (2013) describe effectors from plant parasitic nematodes that target host nuclei and possibly interact with nuclear proteins to establish feeding cells in infected plants. In their Original Research article, Ma et al. (2013) show that nuclear localization of the Avr2 effector from the xylem-colonizing fungus Fusarium oxysporum is required to trigger I2-mediated resistance in tomato plants, whereas Stam et al. (2013) show the diversity of nuclear functions of CRN effectors from the oomycete Phytophthora infestans. Finally, the Opinion article by Noel et al. (2013) discusses recent advances in predicting target sequences of nuclear-targeted TAL effectors from the plant pathogenic bacteria of the genus Xanthomonas. It has been estimated that about 25% of Arabidopsis genes are transcriptionally regulated in response to pathogen infection and a significant number of transcription factors are involved in the defense gene regulation. Raffaele and Rivas (2013) review our current knowledge of the transcriptional control of plant defenses with a focus on the MYB family of transcription factors and, within this family, the Arabidopsis MYB protein AtMYB30, which is a positive regulator of disease resistance. The Review article by Gimenez-Ibanez and Solano (2013) discusses the nuclear crosstalk of jasmonate and salicylate signaling with other hormone pathways during the fine-tuning of a robust plant defence response. Spatial restriction of immune receptors and defense regulators by the nuclear envelope as well as their stimulus-induced nuclear translocation provide an important mechanism for defense regulation, as their level of nuclear accumulation determines the magnitude of the defense response. In addition, nuclear translocation of effectors may also affect subcellular localization of their cognate resistance proteins in a process that is essential for plant immunity. Bhattacharjee et al. (2013) review nuclear functions of different immune receptors and associated proteins, including transcription factors and defence regulators. Chang et al. (2013) discuss how nucleo-cytoplasmic partitioning of the barley MLA immune receptor triggers downstream transcriptional responses, thereby providing an efficient connection between pathogen perception and the plant immune response. Finally, in their Original Research Article, Heidrich et al. (2013) provide evidence that the Arabidopsis WRKY domain-containing immune receptor RRS1 contributes to temperature-conditioned autoimmune responses conferred by a second nuclear immune receptor, RPS4. These data suggest that RPS4 engages RRS1 to direct defence signaling. In summary, recent findings from our rapidly evolving field situate the nucleus at the forefront of the mutual recognition between plants and pathogens. Integrating the knowledge on immunity-associated nuclear events within the outlook of whole cellular dynamics represents an exciting perspective for future research.

The Irish Potato Famine Pathogen Phytophthora infestans Translocates the CRN8 Kinase into Host Plant Cells

Phytopathogenic oomycetes, such as Phytophthora infestans, secrete an arsenal of effector proteins that modulate plant innate immunity to enable infection. We describe CRN8, a host-translocated effector of P. infestans that has kinase activity in planta. CRN8 is a modular protein of the CRN effector family. The C-terminus of CRN8 localizes to the host nucleus and triggers cell death when the protein is expressed in planta. Cell death induction by CRN8 is dependent on its localization to the plant nucleus, which requires a functional nuclear localization signal (NLS). The C-terminal sequence of CRN8 has similarity to a serine/threonine RD kinase domain. We demonstrated that CRN8 is a functional RD kinase and that its auto-phosphorylation is dependent on an intact catalytic site. Co-immunoprecipitation experiments revealed that CRN8 forms a dimer or multimer. Heterologous expression of CRN8 in planta resulted in enhanced virulence by P. infestans. In contrast, in planta expression of the dominant-negative CRN8R469A;D470A resulted in reduced P. infestans infection, further implicating CRN8 in virulence. Overall, our results indicate that similar to animal parasites, plant pathogens also translocate biochemically active kinase effectors inside host cells.