Subfunctionalization of NRC3 altered the genetic structure of the Nicotiana NRC network.

Nucleotide-binding domain and leucine-rich repeat (NLR) proteins play crucial roles in immunity against pathogens in both animals and plants. In solanaceous plants, activation of several sensor NLRs triggers their helper NLRs, known as NLR-required for cell death (NRC), to form resistosome complexes to initiate immune responses. While the sensor NLRs and downstream NRC helpers display diverse genetic compatibility, molecular evolutionary events leading to the complex network architecture remained elusive. Here, we showed that solanaceous NRC3 variants underwent subfunctionalization after the divergence of Solanum and Nicotiana, altering the genetic architecture of the NRC network in Nicotiana. Natural solanaceous NRC3 variants form three allelic groups displaying distinct compatibilities with the sensor NLR Rpi-blb2. Ancestral sequence reconstruction and analyses of natural and chimeric variants identified six key amino acids involved in sensor-helper compatibility. These residues are positioned on multiple surfaces of the resting NRC3 homodimer, collectively contributing to their compatibility with Rpi-blb2. Upon activation, Rpi-blb2-compatible NRC3 variants form membrane-associated punctate and high molecular weight complexes, and confer resistance to the late blight pathogen Phytophthora infestans. Our findings revealed how mutations in NRC alleles lead to subfunctionalization, altering sensor-helper compatibility and contributing to the increased complexity of the NRC network.

A modified Agrobacterium-mediated transformation for two oomycete pathogens.

Oomycetes are a group of filamentous microorganisms that include some of the biggest threats to food security and natural ecosystems. However, much of the molecular basis of the pathogenesis and the development in these organisms remains to be learned, largely due to shortage of efficient genetic manipulation methods. In this study, we developed modified transformation methods for two important oomycete species, Phytophthora infestans and Plasmopara viticola, that bring destructive damage in agricultural production. As part of the study, we established an improved Agrobacterium-mediated transformation (AMT) method by prokaryotic expression in Agrobacterium tumefaciens of AtVIP1 (VirE2-interacting protein 1), an Arabidopsis bZIP gene required for AMT but absent in oomycetes genomes. Using the new method, we achieved an increment in transformation efficiency in two P. infestans strains. We further obtained a positive GFP transformant of P. viticola using the modified AMT method. By combining this method with the CRISPR/Cas12a genome editing system, we successfully performed targeted mutagenesis and generated loss-of-function mutations in two P. infestans genes. We edited a MADS-box transcription factor-encoding gene and found that a homozygous mutation in MADS-box results in poor sporulation and significantly reduced virulence. Meanwhile, a single-copy avirulence effector-encoding gene Avr8 in P. infestans was targeted and the edited transformants were virulent on potato carrying the cognate resistance gene R8, suggesting that loss of Avr8 led to successful evasion of the host immune response by the pathogen. In summary, this study reports on a modified genetic transformation and genome editing system, providing a potential tool for accelerating molecular genetic studies not only in oomycetes, but also other microorganisms.

The WY Domain of an RxLr Effector Drives Interactions with a Host Target Phosphatase to Mimic Host Regulatory Proteins and Promote Phytophthora infestans Infection.

Plant pathogens manipulate the cellular environment of the host to facilitate infection and colonization that often lead to plant diseases. To accomplish this, many specialized pathogens secrete virulence proteins called effectors into the host cell, which subvert processes such as immune signaling, gene transcription, and host metabolism. Phytophthora infestans, the causative agent of potato late blight, employs an expanded repertoire of RxLR effectors with WY domains to manipulate the host through direct interaction with protein targets. However, our understanding of the molecular mechanisms underlying the interactions between WY effectors and their host targets remains limited. In this study, we performed a structural and biophysical characterization of the P. infestans WY effector Pi04314 in complex with the potato Protein Phosphatase 1-c (PP1c). We elucidate how Pi04314 uses a WY domain and a specialized C-terminal loop carrying a KVxF motif that interact with conserved surfaces on PP1c, known to be used by host regulatory proteins for guiding function. Through biophysical and in planta analyses, we demonstrate that Pi04314 WY or KVxF mutants lose their ability to bind PP1c. The loss of PP1c binding correlates with changes in PP1c nucleolar localization and a decrease in lesion size in plant infection assays. This study provides insights into the manipulation of plant hosts by pathogens, revealing how effectors exploit key regulatory interfaces in host proteins to modify their function and facilitate disease. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Transcription factor binding specificities of the oomycete Phytophthora infestans reflect conserved and divergent evolutionary patterns and predict function.

BACKGROUND: Identifying the DNA-binding specificities of transcription factors (TF) is central to understanding gene networks that regulate growth and development. Such knowledge is lacking in oomycetes, a microbial eukaryotic lineage within the stramenopile group. Oomycetes include many important plant and animal pathogens such as the potato and tomato blight agent Phytophthora infestans, which is a tractable model for studying life-stage differentiation within the group. RESULTS: Mining of the P. infestans genome identified 197 genes encoding proteins belonging to 22 TF families. Their chromosomal distribution was consistent with family expansions through unequal crossing-over, which were likely ancient since each family had similar sizes in most oomycetes. Most TFs exhibited dynamic changes in RNA levels through the P. infestans life cycle. The DNA-binding preferences of 123 proteins were assayed using protein-binding oligonucleotide microarrays, which succeeded with 73 proteins from 14 families. Binding sites predicted for representatives of the families were validated by electrophoretic mobility shift or chromatin immunoprecipitation assays. Consistent with the substantial evolutionary distance of oomycetes from traditional model organisms, only a subset of the DNA-binding preferences resembled those of human or plant orthologs. Phylogenetic analyses of the TF families within P. infestans often discriminated clades with canonical and novel DNA targets. Paralogs with similar binding preferences frequently had distinct patterns of expression suggestive of functional divergence. TFs were predicted to either drive life stage-specific expression or serve as general activators based on the representation of their binding sites within total or developmentally-regulated promoters. This projection was confirmed for one TF using synthetic and mutated promoters fused to reporter genes in vivo. CONCLUSIONS: We established a large dataset of binding specificities for P. infestans TFs, representing the first in the stramenopile group. This resource provides a basis for understanding transcriptional regulation by linking TFs with their targets, which should help delineate the molecular components of processes such as sporulation and host infection. Our work also yielded insight into TF evolution during the eukaryotic radiation, revealing both functional conservation as well as diversification across kingdoms.

Botrytis cinerea detoxifies the sesquiterpenoid phytoalexin rishitin through multiple metabolizing pathways.

Botrytis cinerea is a necrotrophic pathogen that infects across a broad range of plant hosts, including high-impact crop species. Its generalist necrotrophic behavior stems from its ability to detoxify structurally diverse phytoalexins. The current study aims to provide evidence of the ability of B. cinerea to tolerate the sesquiterpenoid phytoalexin rishitin, which is produced by potato and tomato. While the growth of potato pathogens Phytophthora infestans (late blight) and Alternaria solani (early blight) was severely inhibited by rishitin, B. cinerea was tolerant to rishitin. After incubation of rishitin with the mycelia of B. cinerea, it was metabolized to at least six oxidized forms. Structural analysis of these purified rishitin metabolites revealed a variety of oxidative metabolism including hydroxylation at C7 or C12, ketone formation at C5, and dihydroxylation at the 10,11-olefin. Six rishitin metabolites showed reduced toxicity to P. infestans and A. solani, indicating that B. cinerea has at least 5 distinct enzymatic reactions to detoxify rishitin. Four host-specialized phytopathogenic Botrytis species, namely B. elliptica, B. allii, B. squamosa, and B. tulipae also had at least a partial ability to metabolize rishitin as B. cinerea, but their metabolic capacity was significantly weaker than that of B. cinerea. These results suggest that the ability of B. cinerea to rapidly metabolize rishitin through multiple detoxification mechanisms could be critical for its pathogenicity in potato and tomato.

Regressive evolution of an effector following a host jump in the Irish potato famine pathogen lineage.

In order to infect a new host species, the pathogen must evolve to enhance infection and transmission in the novel environment. Although we often think of evolution as a process of accumulation, it is also a process of loss. Here, we document an example of regressive evolution of an effector activity in the Irish potato famine pathogen (Phytophthora infestans) lineage, providing evidence that a key sequence motif in the effector PexRD54 has degenerated following a host jump. We began by looking at PexRD54 and PexRD54-like sequences from across Phytophthora species. We found that PexRD54 emerged in the common ancestor of Phytophthora clade 1b and 1c species, and further sequence analysis showed that a key functional motif, the C-terminal ATG8-interacting motif (AIM), was also acquired at this point in the lineage. A closer analysis showed that the P. mirabilis PexRD54 (PmPexRD54) AIM is atypical, the otherwise-conserved central residue mutated from a glutamate to a lysine. We aimed to determine whether this PmPexRD54 AIM polymorphism represented an adaptation to the Mirabilis jalapa host environment. We began by characterizing the M. jalapa ATG8 family, finding that they have a unique evolutionary history compared to previously characterized ATG8s. Then, using co-immunoprecipitation and isothermal titration calorimetry assays, we showed that both full-length PmPexRD54 and the PmPexRD54 AIM peptide bind weakly to the M. jalapa ATG8s. Through a combination of binding assays and structural modelling, we showed that the identity of the residue at the position of the PmPexRD54 AIM polymorphism can underpin high-affinity binding to plant ATG8s. Finally, we conclude that the functionality of the PexRD54 AIM was lost in the P. mirabilis lineage, perhaps owing to as-yet-unknown selection pressure on this effector in the new host environment.

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.

Potato miR394 targets StA/N-INVE and StLCR to negatively regulate late blight resistance.

MicroRNAs (miRNAs) are small noncoding RNAs in eukaryotes. Plant endogenous miRNAs play pivotal roles in regulating plant development and defense responses. MicroRNA394 (miR394) has been reported to regulate plant development, abiotic stresses and defense responses. Previous reports showed that miR394 responded to P. infestans inoculation in potato, indicating that miR394 may be involved in defense responses. In this study, we further investigated its role in potato defense against P. infestans. Stable expression of miR394 in tobacco and potato enhances the susceptibility to P. infestans, which is accompanied with the reduced accumulation of ROS and down-regulation of the PTI (pattern-triggered immunity) marker genes. Besides well-known target StLCR, miR394 also targets StA/N-INVE, which encodes a chloroplast Alkaline/Neutral Invertases (A/N-INVE). Both StLCR and StA/N-INVE positively regulate late blight resistance, while miR394 degrades them. Interestingly, StA/N-INVE is located in the chloroplast, indicating that miR394 may manipulate chloroplast immunity. Degradation of StA/N-INVE may affect the chloroplast function and hence lead to the compromised ROS (reactive oxygen species) burst and reduced retrograde signaling from the chloroplast to the nucleus and cytoplasm. In summary, this study provides new information that miR394 targets and degrades StA/N-INVE and StLCR, which are positive regulators, to enhance potato susceptibility to P. infestans.

Genetic structure and population diversity of Phytophthora infestans strains in Pacific western Canada.

Late blight caused by Phytophthora infestans is an economically important disease of potato and tomato worldwide. In Canada, an increase in late blight incidence and severity coincided with changes in genetic composition of P. infestans. We monitored late blight incidence on tomato and potato in Pacific western and eastern Canada between 2019 and 2022, identified genotypes of P. infestans, and examined their population genetic diversity. We identified four major existing genotypes US11, US17, US8, and US23 as well as 25 new genotypes. The US11 genotype was dominant in Pacific western Canada, accounting for 59% of the total population. We discovered the US17 genotype for the first time in Canada. We revealed a higher incidence of late blight and quite diverse genotypes of P. infestans in Pacific western Canada than in eastern Canada. We found high genetic diversity of P. infestans population from Pacific western Canada, as evidenced by the high number of multilocus genotypes, high values of genetic diversity indices, and emergence of 25 new genotypes. Considering the number of disease incidence, the detection of diverse known genotypes, the emergence of novel genotypes, and the high number of isolates resistant to metalaxyl-m (95%) from Pacific western Canada, the region could play a role in establishing sexual recombination and diverse populations, which could ultimately pose challenges for late blight management. Therefore, continuous monitoring of P. infestans populations in Pacific western region and across Canada is warranted. KEY POINTS: • Genotypes of P. infestans in Pacific western were quite diverse than in eastern Canada. • We discovered US17 genotype for the first time in Canada and identified 26 novel genotypes. • Approximately 95% of P. infestans isolates were resistant to metalaxyl-m.

Reconstructing the Global Migration History of Phytophthora infestans Toward Colombia.

The evolution of new variants of plant pathogens is one of the biggest challenges to controlling and managing plant diseases. Of the forces driving these evolutionary processes, global migration events are particularly important for widely distributed diseases such as potato late blight, caused by the oomycete Phytophthora infestans. However, little is known about its migration routes outside North America and Europe. This work used genotypic data from population studies to elucidate the migration history originating the Colombian P. infestans population. For this purpose, a dataset of 1,706 P. infestans genotypes was recollected, representing North and South America, Europe, and Asia. Descriptive analysis and historical records from North America and Europe were used to propose three global migration hypotheses, differing on the origin of the disease (Mexico or Peru) and the hypothesis that it returned to South America from Europe. These scenarios were tested using approximate Bayesian computation. According to this analysis, the most probable scenario (posterior probability = 0.631) was the one proposing a Peruvian origin for P. infestans, an initial migration toward Colombia and Mexico, and a later event from Mexico to the United States and then to Europe and Asia, with no return to northern South America. In Colombia, the scenario considering a single migration from Peru and posterior migrations within Colombia was the most probable, with a posterior probability of 0.640. The obtained results support the hypothesis of a Peruvian origin for P. infestans followed by rare colonization events worldwide.

Understanding the Temporal Dynamics of Invasive Late Blight Populations in India for Improved Management Practices.

The microbial oomycete pathogen Phytophthora infestans causes severe epidemics of potato late blight in crops globally. Disease management benefits from an understanding of the diversity of pathogen populations. In this study, we explore the dynamics of P. infestans populations in the late blight-potato agro-ecosystem across the Indian subcontinent. Investigations of the macroecological observations at the field level and microbial ecological principles provided insights into future pathogen behavior. We use a comprehensive simple sequence repeat allele dataset to demonstrate that an invasive clonal lineage called EU_13_A2 has dominated populations over 14 years across India, Bangladesh, and Pakistan. Increasing levels of subclonal variation were tracked over time and space, and, for the first time, populations in Asia were also compared with the source populations from Europe. Within India, a regional pathogen population structure was observed with evidence for local migration, cross-border movement between surrounding countries, and introductions via imports. There was also evidence of genetic drift and between-season transmission of more strongly pathogenic subclones with a complete displacement of some subclonal types. The limited introduction of novel genotypes and the use of resistant potato cultivars could contribute to the dominance of the 13_A2 lineage. The insights will contribute to the management of the pathogen in these key global potato production regions.

Genome-wide identification and expression profiles of the Phytophthora infestans responsive CYPome (cytochrome P450 complement) in Solanum tuberosum.

Cytochrome P450s represent one of the largest protein families across all domains of life. In plants, biotic stress can regulate the expression of some P450 genes. However, the CYPome (cytochrome P450 complement) in Solanum tuberosum and its response to Phytophthora infestans infection remains unrevealed. In this study, 488 P450 genes were identified from potato genome, which can be divided into 41 families and 57 subfamilies. Responding to the infection of P. infestans, 375 potato P450 genes were expressed in late blight resistant or susceptible cultivars. A total of 14 P450 genes were identified as resistant related candidates, and 81 P450 genes were identified as late blight responsive candidates. Several phytohormone biosynthesis, brassinosteroid biosynthesis, and phenylpropanoid biosynthesis involved P450 genes were differentially expressed during the potato-pathogen interactions. This study firstly reported the CYPome in S. tuberosum, and characterized the expression patterns of these P450 genes during the infection of P. infestans.

The Variability of Phytophthora infestans Isolates Collected from Estonian Islands in the Baltic Sea.

Knowledge of a pathogen's genetic variability and population structure is of great importance to effective disease management. In this study, 193 isolates of Phytophthora infestans collected from three Estonian islands were characterized over 3 years using simple sequence repeat (SSR) marker data complemented by information on their mating type and resistance to metalaxyl. In combination with SSR marker data from samples in the neighboring Pskov region of Northwest Russia, the impact of regional and landscape structure on the level of genetic exchange was also examined. Among the 111 P. infestans isolates from Estonian islands, 49 alleles were detected among 12 SSR loci, and 59 SSR multilocus genotypes were found, of which 64% were unique. The genetic variation was higher among years than that among islands, as revealed by the analysis of molecular variance. The frequency of metalaxyl-resistant isolates increased from 9% in 2012 to 30% in 2014, and metalaxyl resistance was most frequent among A1 isolates. The test for isolation by distance among the studied regions was not significant, and coupled with the absence of genetic differentiation, the result revealed gene flow and the absence of local adaptation. The data are consistent with a sexual population in which diversity is driven by an annual germination of soilborne oospores. The absence of shared genotypes over the years has important implications when it comes to the management of diseases. Such population diversity can make it difficult to predict the nature of the outbreak in the coming year as the genetic makeup is different for each year.

Comparative transcriptome profiling of potato cultivars infected by late blight pathogen Phytophthora infestans: Diversity of quantitative and qualitative responses.

The Estonia potato cultivar Ando has shown elevated field resistance to Phytophthora infestans, even after being widely grown for over 40 years. A comprehensive transcriptional analysis was performed using RNA-seq from plant leaf tissues to gain insight into the mechanisms activated for the defense after infection. Pathogen infection in Ando resulted in about 5927 differentially expressed genes (DEGs) compared to 1161 DEGs in the susceptible cultivar Arielle. The expression levels of genes related to plant disease resistance such as serine/threonine kinase activity, signal transduction, plant-pathogen interaction, endocytosis, autophagy, mitogen-activated protein kinase (MAPK), and others were significantly enriched in the upregulated DEGs in Ando, whereas in the susceptible cultivar, only the pathway related to phenylpropanoid biosynthesis was enriched in the upregulated DEGs. However, in response to infection, photosynthesis was deregulated in Ando. Multi-signaling pathways of the salicylic-jasmonic-ethylene biosynthesis pathway were also activated in response to Phytophthora infestans infection.

A Variant of LbCas12a and Elevated Incubation Temperatures Enhance the Rate of Gene Editing in the Oomycete Phytophthora infestans.

CRISPR-Cas editing systems have proved to be powerful tools for functional genomics research, but their effectiveness in many non-model species remains limited. In the potato and tomato pathogen Phytophthora infestans, an editing system was previously developed that expresses the Lachnospiracae bacterium Cas12a endonuclease (LbCas12a) and guide RNA from a DNA vector. However, the method works at low efficiency. Based on a hypothesis that editing is constrained by a mismatch between the optimal temperatures for P. infestans growth and endonuclease catalysis, we tested two strategies that increased the frequency of editing of two target genes by about 10-fold. First, we found that editing was boosted by a mutation in LbCas12a (D156R) that had been reported to expand its catalytic activity over a broader temperature range. Second, we observed that editing was enhanced by transiently incubating transformed tissue at a higher temperature. These modifications should make CRISPR-Cas12a more useful for interrogating gene and protein function in P. infestans and its relatives, especially species that grow optimally at lower temperatures. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

EffectorO: Motif-Independent Prediction of Effectors in Oomycete Genomes Using Machine Learning and Lineage Specificity.

Oomycete plant pathogens cause a wide variety of diseases, including late blight of potato, sudden oak death, and downy mildews of plants. These pathogens are major contributors to loss in numerous food crops. Oomycetes secrete effector proteins to manipulate their hosts to the advantage of the pathogen. Plants have evolved to recognize effectors, resulting in an evolutionary cycle of defense and counter-defense in plant-microbe interactions. This selective pressure results in highly diverse effector sequences that can be difficult to computationally identify using only sequence similarity. We developed a novel effector prediction tool, EffectorO, that uses two complementary approaches to predict effectors in oomycete pathogen genomes: i) a machine learning-based pipeline that predicts effector probability based on the biochemical properties of the N-terminal amino-acid sequence of a protein and ii) a pipeline based on lineage specificity to find proteins that are unique to one species or genus, a sign of evolutionary divergence due to adaptation to the host. We tested EffectorO on Bremia lactucae, which causes lettuce downy mildew, and Phytophthora infestans, which causes late blight of potato and tomato, and predicted many novel effector candidates while recovering the majority of known effector candidates. EffectorO will be useful for discovering novel families of oomycete effectors without relying on sequence similarity to known effectors. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The catalase-peroxidase PiCP1 plays a critical role in abiotic stress resistance, pathogenicity and asexual structure development in Phytophthora infestans.

Catalase-peroxidase is a heme oxidoreductase widely distributed in bacteria and lower eukaryotes. In this study, we identified a catalase-peroxidase PiCP1 (PITG_05579) in Phytophthora infestans. PiCP1 had catalase/peroxidase and secretion activities and was highly expressed in sporangia and upregulated in response to oxidative and heat stresses. Compared with wild type, PiCP1-silenced transformants (STs) had decreased catalase activity, reduced oxidant stress resistance and damped cell wall integrity. In contrast, PiCP1-overexpression transformants (OTs) demonstrated increased tolerance to abiotic stresses and induced the upregulation of PR genes in the host salicylic acid pathway. The high concentration of PiCP1 can also induced callose deposition in plant tissue. Importantly, both STs and OTs have severely reduced sporangia formation and zoospore releasing rate, but the sporangia germination rate and type varied depending on environmental conditions. Comparative sequence analyses show that catalase-peroxidases are broadly distributed and highly conserved among soil-borne plant parasitic oomycetes, but not in freshwater-inhabiting or strictly plants-inhabiting oomycetes. In addition, we found that silencing PiCP1 downregulated the expression of PiCAT2. These results revealed the important roles of PiCP1 in abiotic stress resistance, pathogenicity and in regulating asexual structure development in response to environmental change. Our findings provide new insights into catalase-peroxidase functions in eukaryotic pathogens.

Characterization of host-effector transcription dynamics during pathogen infection in engineered late blight resistant potato.

Phytophthora infestans, the etiologic agent of late blight, is a threat to potato production in areas with high humidity during the growing season. The oomycete pathogen is hemi-biotrophic, it establishes infection on living plant cells and then spreads, kills, and feeds off the necrotized plant tissue material. The interaction between host and pathogen is complex with dynamic pathogen RXLR effectors and potato NB-LRR resistance proteins actively competing for dominance and survival. Late blight protection was brought to several cultivars of potato through insertion of the wild potato (Solanum venturii) NB-LRR resistance gene Rpi-vnt1.1. We have established that the late blight protection trait, mediated by Rpi-vnt1.1, is effective despite low expression of RNA. The RNA expression dynamics of Rpi-vnt1.1 and the cognate pathogen RXLR effector, Avr-vnt1, were evaluated following spray inoculation with up to five different contemporary late blight isolates from North America and South America. Following inoculations, RXLR effector transcript profiles provided insight into interaction compatibility in relation to markers of the late blight hemi-biotrophic lifecycle.

Variation in the Outcome of Plant-Mediated Pathogen Interactions in Potato: Effects of Initial Infections on Conspecific vs. Heterospecific Subsequent Infections.

Plants are often attacked sequentially by multiple enemies. Pathogen sequential co-infections can lead to indirect interactions mediated by plant induced responses whose outcome is contingent on differences in the magnitude and type of plant induced defences elicited by different species or guilds. To date, however, most studies have tested unidirectional effects of one pathogen on another, not discerning between conspecific vs. heterospecific infections, and often not measuring plant induced responses underlying such outcomes. To address this, we conducted a greenhouse experiment testing for the impact of initial infection by two leaf pathogens (Alternaria solani and Phytophthora infestans) on subsequent infection by each of these pathogens on potato (Solanum tuberosum) plants, and also measured induced plant defences (phenolic compounds) to inform on interaction outcomes. We found contrasting results depending on the identity of the initially infecting pathogen. Specifically, initial infection by A. solani drove induced resistance (lower necrosis) by subsequently infecting A. solani (conspecific induced resistance) but had no effect on subsequent infection by P. infestans. In contrast, initial infection by P. infestans drove induced resistance to subsequent infection by both conspecifics and A. solani. Patterns of plant induced defences correlated with (and potentially explained) induced resistance to conspecific but not heterospecific (e.g., in the case of P. infestans) subsequent infection. Overall, these results further our understanding of plant-mediated pathogen interactions by showing that plant-mediated interactions between pathogen species can be asymmetrical and in some cases not reciprocal, that pathogen species can vary in the importance of conspecific vs. heterospecific effects, and shed mechanistic insight into the role of plant induced responses driving such interactions.

MicroRNA Expression Profiles in Response to Phytophthora infestans and Oidium neolycopersici and Functional Identification of sly-miR397 in Tomato.

Late blight and powdery mildew are two widespread tomato diseases caused by Phytophthora infestans and Oidium neolycopersici, respectively, which reduce the quantity and quality of tomato. MicroRNAs (miRNAs) play critical roles in tomato resistance to various pathogens. Investigating the function of miRNAs is of great significance in controlling tomato diseases. To identify potential miRNAs involved in the interaction of tomato with P. infestans or O. neolycopersici, we analyzed the expression profiles of small RNAs in tomato leaves infected with these two pathogens using RNA-seq technology. A total of 330 and 288 miRNAs exhibited differences in expression levels after exposure to P. infestans and O. neolycopersici, respectively. One hundred and forty-six commonly differentially expressed (DE) miRNAs responsive to P. infestans and O. neolycopersici infestation were detected, including 10 commonly known conserved DE miRNAs and 136 novel miRNAs. Among these known DE miRNAs, sly-miR397 was strongly downregulated in response to P. infestans or O. neolycopersici infection. Silencing of sly-miR397 resulted in enhanced tolerance to the pathogens, whereas overexpression of sly-miR397 showed increased susceptibility. Furthermore, changes in sly-miR397 expression could also affect expression levels of pathogenesis-related genes and reactive oxygen species-scavenging genes, leading to altered necrotic cells and H2O2 levels. In addition, the number of lateral branches significantly changed in transgenic plants. Taken together, our results provide potential miRNA resources for further research of miRNA-disease associations and indicates that sly-miR397 acts as a negative regulator of disease resistance and influences lateral branch development in tomato.