Network analyses predict major regulators of resistance to early blight disease complex in tomato.

BACKGROUND: Early blight and brown leaf spot are often cited as the most problematic pathogens of tomato in many agricultural regions. Their causal agents are Alternaria spp., a genus of Ascomycota containing numerous necrotrophic pathogens. Breeding programs have yielded quantitatively resistant commercial cultivars, but fungicide application remains necessary to mitigate the yield losses. A major hindrance to resistance breeding is the complexity of the genetic determinants of resistance and susceptibility. In the absence of sufficiently resistant germplasm, we sequenced the transcriptomes of Heinz 1706 tomatoes treated with strongly virulent and weakly virulent isolates of Alternaria spp. 3 h post infection. We expanded existing functional gene annotations in tomato and using network statistics, we analyzed the transcriptional modules associated with defense and susceptibility. RESULTS: The induced responses are very distinct. The weakly virulent isolate induced a defense response of calcium-signaling, hormone responses, and transcription factors. These defense-associated processes were found in a single transcriptional module alongside secondary metabolite biosynthesis genes, and other defense responses. Co-expression and gene regulatory networks independently predicted several D clade ethylene response factors to be early regulators of the defense transcriptional module, as well as other transcription factors both known and novel in pathogen defense, including several JA-associated genes. In contrast, the strongly virulent isolate elicited a much weaker response, and a separate transcriptional module bereft of hormone signaling. CONCLUSIONS: Our findings have predicted major defense regulators and several targets for downstream functional analyses. Combined with our improved gene functional annotation, they suggest that defense is achieved through induction of Alternaria-specific immune pathways, and susceptibility is mediated by modulating hormone responses. The implication of multiple specific clade D ethylene response factors and upregulation of JA-associated genes suggests that host defense in this pathosystem involves ethylene response factors to modulate jasmonic acid signaling.

Untargeted metabolomics reveals PTI-associated metabolites.

Plants employ a multilayered immune system to combat pathogens. In one layer, recognition of Pathogen- or Microbe-Associated Molecular Patterns or elicitors, triggers a cascade that leads to defence against the pathogen and Pattern Triggered Immunity. Secondary or specialised metabolites (SMs) are expected to play a role, because they are potentially anti-fungal compounds. Tomato (Solanum lycopersicum) plants inoculated with Alternaria solani s.l. show symptoms of infection after inoculation. Plants inoculated with Alternaria alternata remain symptomless. We hypothesised that pattern-triggered induction of resistance related metabolites in tomato contributes to the resistance against A. alternata. We compared the metabolomic profile (metabolome) of tomato after treatments with A. alternata, A. solani and the fungal elicitor chitin, and identified SMs involved in early defence of tomato plants. We revealed differential metabolome fingerprints. The composition of A. alternata and chitin induced metabolomes show larger overlap with each other than with the A. solani induced metabolome. We identify 65 metabolites possibly associated with PTI in tomato plants, including NAD and trigonelline. We confirm that trigonelline inhibits fungal growth in vitro at physiological concentrations. Thus, a true pattern-triggered, chemical defence is mounted against A. alternata, which contains anti-fungal compounds that could be interesting for crop protection strategies.

Small-spored Alternaria spp. (section Alternaria) are common pathogens on wild tomato species.

The wild relatives of modern tomato crops are native to South America. These plants occur in habitats as different as the Andes and the Atacama Desert and are, to some degree, all susceptible to fungal pathogens of the genus Alternaria. Alternaria is a large genus. On tomatoes, several species cause early blight, leaf spots and other diseases. We collected Alternaria-like infection lesions from the leaves of eight wild tomato species from Chile and Peru. Using molecular barcoding markers, we characterized the pathogens. The infection lesions were caused predominantly by small-spored species of Alternaria of the section Alternaria, like A. alternata, but also by Stemphylium spp., Alternaria spp. from the section Ulocladioides and other related species. Morphological observations and an infection assay confirmed this. Comparative genetic diversity analyses show a larger diversity in this wild system than in studies of cultivated Solanum species. As A. alternata has been reported to be an increasing problem in cultivated tomatoes, investigating the evolutionary potential of this pathogen is not only interesting to scientists studying wild plant pathosystems. It could also inform crop protection and breeding programs to be aware of potential epidemics caused by species still confined to South America.

Laminarin-triggered defence responses are geographically dependent in natural populations of Solanum chilense.

Natural plant populations are polymorphic and show intraspecific variation in resistance properties against pathogens. The activation of the underlying defence responses can depend on variation in perception of pathogen-associated molecular patterns or elicitors. To dissect such variation, we evaluated the responses induced by laminarin (a glucan, representing an elicitor from oomycetes) in the wild tomato species Solanum chilense and correlated this to observed infection frequencies of Phytophthora infestans. We measured reactive oxygen species burst and levels of diverse phytohormones upon elicitation in 83 plants originating from nine populations. We found high diversity in basal and elicitor-induced levels of each component. Further we generated linear models to explain the observed infection frequency of P. infestans. The effect of individual components differed dependent on the geographical origin of the plants. We found that the resistance in the southern coastal region, but not in the other regions, was directly correlated to ethylene responses and confirmed this positive correlation using ethylene inhibition assays. Our findings reveal high diversity in the strength of defence responses within a species and the involvement of different components with a quantitatively different contribution of individual components to resistance in geographically separated populations of a wild plant species.

Barley shows reduced Fusarium head blight under drought and modular expression of differentially expressed genes under combined stress.

Plants often face simultaneous abiotic and biotic stress conditions; however, physiological and transcriptional responses under such combined stress conditions are still not fully understood. Spring barley (Hordeum vulgare) is susceptible to Fusarium head blight (FHB), which is strongly affected by weather conditions. We therefore studied the potential influence of drought on FHB severity and plant responses in three varieties of different susceptibility. We found strongly reduced FHB severity in susceptible varieties under drought. The number of differentially expressed genes (DEGs) and strength of transcriptomic regulation reflected the concentrations of physiological stress markers such as abscisic acid or fungal DNA contents. Infection-related gene expression was associated with susceptibility rather than resistance. Weighted gene co-expression network analysis revealed 18 modules of co-expressed genes that reflected the pathogen- or drought-response in the three varieties. A generally infection-related module contained co-expressed genes for defence, programmed cell death, and mycotoxin detoxification, indicating that the diverse genotypes used a similar defence strategy towards FHB, albeit with different degrees of success. Further, DEGs showed co-expression in drought- or genotype-associated modules that correlated with measured phytohormones or the osmolyte proline. The combination of drought stress with infection led to the highest numbers of DEGs and resulted in a modular composition of the single-stress responses rather than a specific transcriptional output.

Distinct Phyllosphere Microbiome of Wild Tomato Species in Central Peru upon Dysbiosis.

Plants are colonized by myriads of microbes across kingdoms, which affect host development, fitness, and reproduction. Hence, plant microbiomes have been explored across a broad range of host species, including model organisms, crops, and trees under controlled and natural conditions. Tomato is one of the world's most important vegetable crops; however, little is known about the microbiota of wild tomato species. To obtain insights into the tomato microbiota occurring in natural environments, we sampled epiphytic microbes from leaves of four tomato species, Solanum habrochaites, S. corneliomulleri, S. peruvianum, and S. pimpinellifolium, from two geographical locations within the Lima region of Peru over 2 consecutive years. Here, a high-throughput sequencing approach was applied to investigate microbial compositions including bacteria, fungi, and eukaryotes across tomato species and geographical locations. The phyllosphere microbiome composition varies between hosts and location. Yet, we identified persistent microbes across tomato species that form the tomato microbial core community. In addition, we phenotypically defined healthy and dysbiotic samples and performed a downstream analysis to reveal the impact on microbial community structures. To do so, we compared microbial diversities, unique OTUs, relative abundances of core taxa, and microbial hub taxa, as well as co-occurrence network characteristics in healthy and dysbiotic tomato leaves and found that dysbiosis affects the phyllosphere microbial composition in a host species-dependent manner. Yet, overall, the present data suggests an enrichment of plant-promoting microbial taxa in healthy leaves, whereas numerous microbial taxa containing plant pathogens occurred in dysbiotic leaves.Concluding, we identify the core phyllosphere microbiome of wild tomato species, and show that the overall phyllosphere microbiome can be impacted by sampling time point, geographical location, host genotype, and plant health. Future studies in these components will help understand the microbial contribution to plant health in natural systems and can be of use in cultivated tomatoes.

Metabolomics in Plant Pathogen Defense: From Single Molecules to Large-Scale Analysis.

Plants produce a high diversity of secondary metabolites that are involved in a wide range of different functions, including stress tolerance, signaling molecules for interactions with other species (allelopathy), and protecting plants against herbivores and pathogens. With the rise of more accessible, high-throughput mass spectrometry and new analytical tools, it becomes feasible to identify and validate new secondary metabolites involved in pathogen resistance or assign new roles to previously detected compounds. In this review, we provide a brief overview of the major pathogen defense-associated classes of secondary metabolites, with a focus on those with direct anti-pathogen function. For each class, we highlight one or more typical examples representing the class to give a comprehensive summary of some of the work done to date. In the second part of this review, we highlight how new technological advances and high-throughput experiments in combination with other sources of -omics data, such as genomics and transcriptomics, can accelerate the studies on secondary metabolites and help to link these compounds to genotypes. Employing such approaches will improve our understanding of chemical defenses against plant pathogens and allow for rapid development of markers for resistance breeding.

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

Phytophthora infestans is a pathogenic oomycete that causes the infamous potato late blight disease. Resistance (R) genes from diverse Solanum species encode intracellular receptors that trigger effective defense responses upon the recognition of cognate RXLR avirulence (Avr) effector proteins. 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. In this study, we cloned 16 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 effector recognition and co-evolution. Functional and non-functional alleles of Rpi-chc1 encode coiled-coil nucleotide-binding leucine-rich repeat (CNL) proteins, being the first described representatives of the CNL16 family. These alleles have distinct patterns of RXLR effector recognition. While Rpi-chc1.1 recognized multiple PexRD12 (Avrchc1.1) proteins, Rpi-chc1.2 recognized multiple PexRD31 (Avrchc1.2) proteins, both belonging to the PexRD12/31 effector superfamily. Domain swaps between Rpi-chc1.1 and Rpi-chc1.2 revealed that overlapping subdomains in the leucine-rich repeat (LRR) domain are 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 guide future strategies to breed durable resistant varieties.

A Conserved Oomycete CRN Effector Targets Tomato TCP14-2 to Enhance Virulence.

Phytophthora spp. secrete vast arrays of effector molecules during infection to aid in host colonization. The crinkling and necrosis (CRN) protein family forms an extensive repertoire of candidate effectors that accumulate in the host nucleus to perturb processes required for immunity. Here, we show that CRN12_997 from Phytophthora capsici binds a TCP transcription factor, SlTCP14-2, to inhibit its immunity-associated activity against Phytophthora spp. Coimmunoprecipitation and bimolecular fluorescence complementation studies confirm a specific CRN12_997-SlTCP14-2 interaction in vivo. Coexpression of CRN12_997 specifically counteracts the TCP14-enhanced immunity phenotype, suggesting that CRN mediated perturbation of SlTCP14-2 function. We show that SlTCP14-2 associates with nuclear chromatin and that CRN12_997 diminishes SlTCP14-2 DNA binding. Collectively, our data support a model in which SlTCP14-2 associates with chromatin to enhance immunity. The interaction between CRN12_997 and SlTCP14-2 reduces DNA binding of the immune regulator. We propose that the modulation of SlTCP14-2 chromatin affinity, caused by CRN12-997, enhances susceptibility to P. capsici.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

ONT-Based Draft Genome Assembly and Annotation of Alternaria atra.

Species of Alternaria (phylum Ascomycota, family Pleosporaceae) are known as serious plant pathogens, causing major losses on a wide range of crops. Alternaria atra (previously known as Ulocladium atrum) can grow as a saprophyte on many hosts and causes Ulocladium blight on potato. It has been reported that it can also be used as a biocontrol agent against Botrytis cinerea. Here, we present a scaffold-level reference genome assembly for A. atra. The assembly contains 43 scaffolds with a total length of 39.62 Mbp, with scaffold N50 of 3,893,166 bp, L50 of 4, and the longest 10 scaffolds containing 89.9% of the assembled data. RNA-sequencing-guided gene prediction using BRAKER resulted in 12,173 protein-coding genes with their functional annotation. This first high-quality reference genome assembly and annotation for A. atra can be used as a resource for studying evolution in the highly complicated Alternaria genus and might help in understanding the mechanisms defining its role as pathogen or biocontrol agent.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Population Genomics of Filamentous Plant Pathogens-A Brief Overview of Research Questions, Approaches, and Pitfalls.

With ever-decreasing sequencing costs, research on the population biology of plant pathogens is transitioning from population genetics-using dozens of genetic markers or polymorphism data of several genes-to population genomics-using several hundred to tens of thousands of markers or whole-genome sequence data. The field of population genomics is characterized by rapid theoretical and methodological advances and by numerous steps and pitfalls in its technical and analytical workflow. In this article, we aim to provide a brief overview of topics relevant to the study of population genomics of filamentous plant pathogens and direct readers to more extensive reviews for in-depth understanding. We briefly discuss different types of population genomics-inspired research questions and give insights into the sampling strategies that can be used to answer such questions. We then consider different sequencing strategies, the various options available for data processing, and some of the currently available tools for population genomic data analysis. We conclude by highlighting some of the hurdles along the population genomic workflow, providing cautionary warnings relative to assumptions and technical challenges, and presenting our own future perspectives of the field of population genomics for filamentous plant pathogens.

Population Genomic- and Phylogenomic-Enabled Advances to Increase Insight Into Pathogen Biology and Epidemiology.

Population genetics has been a key discipline in phytopathology for many years. The recent rise in cost-effective, high-throughput DNA sequencing technologies, allows sequencing of dozens, if not hundreds of specimens, turning population genetics into population genomics and opening up new, exciting opportunities as described in this Focus Issue. Without the limitations of genetic markers and the availability of whole or near whole-genome data, population genomics can give new insights into the biology, evolution and adaptation, and dissemination patterns of plant-associated microbes.

Population studies of the wild tomato species Solanum chilense reveal geographically structured major gene-mediated pathogen resistance.

Natural plant populations encounter strong pathogen pressure and defence-associated genes are known to be under selection dependent on the pressure by the pathogens. Here, we use populations of the wild tomato Solanum chilense to investigate natural resistance against Cladosporium fulvum, a well-known ascomycete pathogen of domesticated tomatoes. Host populations used are from distinct geographical origins and share a defined evolutionary history. We show that distinct populations of S. chilense differ in resistance against the pathogen. Screening for major resistance gene-mediated pathogen recognition throughout the whole species showed clear geographical differences between populations and complete loss of pathogen recognition in the south of the species range. In addition, we observed high complexity in a homologues of Cladosporium resistance (Hcr) locus, underlying the recognition of C. fulvum, in central and northern populations. Our findings show that major gene-mediated recognition specificity is diverse in a natural plant-pathosystem. We place major gene resistance in a geographical context that also defined the evolutionary history of that species. Data suggest that the underlying loci are more complex than previously anticipated, with small-scale gene recombination being possibly responsible for maintaining balanced polymorphisms in the populations that experience pathogen pressure.

Subsets of NLR genes show differential signatures of adaptation during colonization of new habitats.

Nucleotide binding site, leucine-rich repeat receptors (NLRs) are canonical resistance (R) genes in plants, fungi and animals, functioning as central (helper) and peripheral (sensor) genes in a signalling network. We investigate NLR evolution during the colonization of novel habitats in a model tomato species, Solanum chilense. We used R-gene enrichment sequencing to obtain polymorphism data at NLRs of 140 plants sampled across 14 populations covering the whole species range. We inferred the past demographic history of habitat colonization by resequencing whole genomes from three S. chilense plants from three key populations and performing approximate Bayesian computation using data from the 14 populations. Using these parameters, we simulated the genetic differentiation statistics distribution expected under neutral NLR evolution and identified small subsets of outlier NLRs exhibiting signatures of selection across populations. NLRs under selection between habitats are more often helper genes, whereas those showing signatures of adaptation in single populations are more often sensor-NLRs. Thus, centrality in the NLR network does not constrain NLR evolvability, and new mutations in central genes in the network are key for R-gene adaptation during colonization of different habitats.

The Current Epidemic of the Barley Pathogen Ramularia collo-cygni Derives from a Population Expansion and Shows Global Admixture.

Ramularia leaf spot is becoming an ever-increasing problem in main barley-growing regions since the 1980s, causing up to 70% yield loss in extreme cases. Yet, the causal agent Ramularia collo-cygni remains poorly studied. The diversity of the pathogen in the field thus far remains unknown. Furthermore, it is unknown to what extent the pathogen has a sexual reproductive cycle. The teleomorph of R. collo-cygni has not been observed. To study the genetic diversity of R. collo-cygni and get more insights in its biology, we sequenced the genomes of 19 R. collo-cygni isolates from multiple geographic locations and diverse hosts. Nucleotide polymorphism analyses of all isolates shows that R. collo-cygni is genetically diverse worldwide, with little geographic or host specific differentiation. Next, we used two different methods to detect signals of recombination in our sample set. Both methods find putative recombination events, which indicate that sexual reproduction happens or has happened in the global R. collo-cygni population. Lastly, we used these data on recombination to perform historic population size analyses. These suggest that the effective population size of R. collo-cygni decreased during the domestication of barley and subsequently grew with the rise of agriculture. Our findings deepen our understanding of R. collo-cygni biology and can help us to understand the current epidemic. We discuss how our findings support possible global spread through seed transfer, and we highlight how recombination, clonal spreading, and lack of host specificity could amplify global epidemics of this increasingly important disease and suggest specific approaches to combat the pathogen.

An NMRA-Like Protein Regulates Gene Expression in Phytophthora capsici to Drive the Infection Cycle on Tomato.

Phytophthora spp. cause devastating disease epidemics on important crop plants and pose a grave threat to global crop production. Critically, Phytophthora pathogens represent a distinct evolutionary lineage in which pathogenicity has been acquired independently. Therefore, there is an urgent need to understand and disrupt the processes that drive infection if we aspire to defeat oomycete pathogens in the field. One area that has received little attention thus far in this respect is the regulation of Phytophthora gene expression during infection. Here, we characterize PcNMRAL1 (Phyca11_505845), a homolog of the Aspergillus nidulans nitrogen metabolite repression regulator NMRA and demonstrate a role for this protein in progression of the Phytophthora capsici infection cycle. PcNmrAL1 is coexpressed with the biotrophic marker gene PcHmp1 (haustorial membrane protein 1) and, when overexpressed, extends the biotrophic infection stage. Microarray analyses revealed that PcNmrAL1 overexpression in P. capsici leads to large-scale transcriptional changes during infection and in vitro. Importantly, detailed analysis reveals that PcNmrAL1 overexpression induces biotrophy-associated genes while repressing those associated with necrotrophy. In addition to factors controlling transcription, translation, and nitrogen metabolism, PcNMRAL1 helps regulate the expression of a considerable effector repertoire in P. capsici. Our data suggests that PcNMRAL1 is a transcriptional regulator that mediates the biotrophy to necrotrophy transition. PcNMRAL1 represents a novel factor that may drive the Phytophthora disease cycle on crops. This study provides the first insight into mechanisms that regulate infection-related processes in Phytophthora spp. and provides a platform for further studies aimed at disabling pathogenesis and preventing crop losses.

Diversification of defensins and NLRs in Arabidopsis species by different evolutionary mechanisms.

BACKGROUND: Genes encoding proteins underlying host-pathogen co-evolution and which are selected for new resistance specificities frequently are under positive selection, a process that maintains diversity. Here, we tested the contribution of natural selection, recombination and transcriptional divergence to the evolutionary diversification of the plant defensins superfamily in three Arabidopsis species. The intracellular NOD-like receptor (NLR) family was used for comparison because positive selection has been well documented in its members. Similar to defensins, NLRs are encoded by a large and polymorphic gene family and many of their members are involved in the immune response. RESULTS: Gene trees of Arabidopsis defensins (DEFLs) show a high prevalence of clades containing orthologs. This indicates that their diversity dates back to a common ancestor and species-specific duplications did not significantly contribute to gene family expansion. DEFLs are characterized by a pervasive pattern of neutral evolution with infrequent positive and negative selection as well as recombination. In comparison, most NLR alignment groups are characterized by frequent occurrence of positive selection and recombination in their leucine-rich repeat (LRR) domain as well negative selection in their nucleotide-binding (NB-ARC) domain. While major NLR subgroups are expressed in pistils and leaves both in presence or absence of pathogen infection, the members of DEFL alignment groups are predominantly transcribed in pistils. Furthermore, conserved groups of NLRs and DEFLs are differentially expressed in response to Fusarium graminearum regardless of whether these genes are under positive selection or not. CONCLUSIONS: The present analyses of NLRs expands previous studies in Arabidopsis thaliana and highlights contrasting patterns of purifying and diversifying selection affecting different gene regions. DEFL genes show a different evolutionary trend, with fewer recombination events and significantly fewer instances of natural selection. Their heterogeneous expression pattern suggests that transcriptional divergence probably made the major contribution to functional diversification. In comparison to smaller families encoding pathogenesis-related (PR) proteins under positive selection, DEFLs are involved in a wide variety of processes that altogether might pose structural and functional trade-offs to their family-wide pattern of evolution.

Quantitative analysis of the tomato nuclear proteome during Phytophthora capsici infection unveils regulators of immunity.

Plant-pathogen interactions are complex associations driven by the interplay of host and microbe-encoded factors. With secreted pathogen proteins (effectors) and immune signalling components found in the plant nucleus, this compartment is a battleground where susceptibility is specified. We hypothesized that, by defining changes in the nuclear proteome during infection, we can pinpoint vital components required for immunity or susceptibility. We tested this hypothesis by documenting dynamic changes in the tomato (Solanum lycopersicum) nuclear proteome during infection by the oomycete pathogen Phytophthora capsici. We enriched nuclei from infected and noninfected tissues and quantitatively assessed changes in the nuclear proteome. We then tested the role of candidate regulators in immunity through functional assays. We demonstrated that the host nuclear proteome dynamically changes during P. capsici infection. We observed that known nuclear immunity factors were differentially expressed and, based on this observation, selected a set of candidate regulators that we successfully implicated in immunity to P. capsici. Our work exemplifies a powerful strategy to gain rapid insight into important nuclear processes that underpin complex crop traits such as resistance. We have identified a large set of candidate nuclear factors that may underpin immunity to pathogens in crops.

Mp10 and Mp42 from the aphid species Myzus persicae trigger plant defenses in Nicotiana benthamiana through different activities.

Aphids are phloem-feeding insects that, like other plant parasites, deliver effectors inside their host to manipulate host responses. The Myzus persicae (green peach aphid) candidate effectors Mp10 and Mp42 were previously found to reduce aphid fecundity upon intracellular transient overexpression in Nicotiana benthamiana. We performed functional analyses of these proteins to investigate whether they activate defenses through similar activities. We employed a range of functional characterization experiments based on intracellular transient overexpression in N. benthamiana to determine the subcellular localization of Mp10 and Mp42 and investigate their role in activating plant defense signaling. Mp10 and Mp42 showed distinct subcellular localization in planta, suggesting that they target different host compartments. Also, Mp10 reduced the levels of Agrobacterium-mediated overexpression of proteins. This reduction was not due to an effect on Agrobacterium viability. Transient overexpression of Mp10 but not Mp42 activated jasmonic acid and salicylic acid signaling pathways and decreased susceptibility to the hemibiotrophic plant pathogen Phytophthora capsici. We found that two candidate effectors from the broad-host-range aphid M. persicae can trigger aphid defenses through different mechanisms. Importantly, we found that some (candidate) effectors such as Mp10 interfere with Agrobacterium-based overexpression assays, an important tool to study effector activity and function.