Comparative analysis of soybean transcriptional profiles reveals defense mechanisms involved in resistance against Diaporthe caulivora.

Soybean stem canker (SSC) caused by the fungal pathogen Diaporthe caulivora is an important disease affecting soybean production worldwide. However, limited information related to the molecular mechanisms underlying soybean resistance to Diaporthe species is available. In the present work, we analyzed the defense responses to D. caulivora in the soybean genotypes Williams and Génesis 5601. The results showed that compared to Williams, Génesis 5601 is more resistant to fungal infection evidenced by significantly smaller lesion length, reduced disease severity and pathogen biomass. Transcriptional profiling was performed in untreated plants and in D. caulivora-inoculated and control-treated tissues at 8 and 48 h post inoculation (hpi). In total, 2.322 and 1.855 genes were differentially expressed in Génesis 5601 and Williams, respectively. Interestingly, Génesis 5601 exhibited a significantly higher number of upregulated genes compared to Williams at 8 hpi, 1.028 versus 434 genes. Resistance to D. caulivora was associated with defense activation through transcriptional reprogramming mediating perception of the pathogen by receptors, biosynthesis of phenylpropanoids, hormone signaling, small heat shock proteins and pathogenesis related (PR) genes. These findings provide novel insights into soybean defense mechanisms leading to host resistance against D. caulivora, and generate a foundation for the development of resistant SSC varieties within soybean breeding programs.

Comparative genomics of plant pathogenic Diaporthe species and transcriptomics of Diaporthe caulivora during host infection reveal insights into pathogenic strategies of the genus.

BACKGROUND: Diaporthe caulivora is a fungal pathogen causing stem canker in soybean worldwide. The generation of genomic and transcriptomic information of this ascomycete, together with a comparative genomic approach with other pathogens of this genus, will contribute to get insights into the molecular basis of pathogenicity strategies used by D. caulivora and other Diaporthe species. RESULTS: In the present work, the nuclear genome of D. caulivora isolate (D57) was resolved, and a comprehensive annotation based on gene expression and genomic analysis is provided. Diaporthe caulivora D57 has an estimated size of 57,86 Mb and contains 18,385 predicted protein-coding genes, from which 1501 encode predicted secreted proteins. A large array of D. caulivora genes encoding secreted pathogenicity-related proteins was identified, including carbohydrate-active enzymes (CAZymes), necrosis-inducing proteins, oxidoreductases, proteases and effector candidates. Comparative genomics with other plant pathogenic Diaporthe species revealed a core secretome present in all Diaporthe species as well as Diaporthe-specific and D. caulivora-specific secreted proteins. Transcriptional profiling during early soybean infection stages showed differential expression of 2659 D. caulivora genes. Expression patterns of upregulated genes and gene ontology enrichment analysis revealed that host infection strategies depends on plant cell wall degradation and modification, detoxification of compounds, transporter activities and toxin production. Increased expression of effectors candidates suggests that D. caulivora pathogenicity also rely on plant defense evasion. A high proportion of the upregulated genes correspond to the core secretome and are represented in the pathogen-host interaction (PHI) database, which is consistent with their potential roles in pathogenic strategies of the genus Diaporthe. CONCLUSIONS: Our findings give novel and relevant insights into the molecular traits involved in pathogenicity of D. caulivora towards soybean plants. Some of these traits are in common with other Diaporthe pathogens with different host specificity, while others are species-specific. Our analyses also highlight the importance to have a deeper understanding of pathogenicity functions among Diaporthe pathogens and their interference with plant defense activation.

Soybean Stem Canker Caused by Diaporthe caulivora; Pathogen Diversity, Colonization Process, and Plant Defense Activation.

Soybean is an important crop in South America, and its production is limited by fungal diseases caused by species from the genus Diaporthe, including seed decay, pod and stem blight, and soybean stem canker (SSC). In this study, we focused on Diaporthe species isolated from soybean plants with SSC lesions in different parts of Uruguay. Diaporthe diversity was determined by sequencing the internal transcribed spacer (ITS) regions of ribosomal RNA and a partial region of the translation elongation factor 1-alpha gene (TEF1α). Phylogenetic analysis showed that the isolates belong to five defined groups of Diaporthe species, Diaporthe caulivora and Diaporthe longicolla being the most predominant species present in stem canker lesions. Due to the importance of D. caulivora as the causal agent of SSC in the region and other parts of the world, we further characterized the interaction of this pathogen with soybean. Based on genetic diversity of D. caulivora isolates evaluated with inter-sequence single repetition (ISSR), three different isolates were selected for pathogenicity assays. Differences in virulence were observed among the selected D. caulivora isolates on susceptible soybean plants. Further inspection of the infection and colonization process showed that D. caulivora hyphae are associated with trichomes in petioles, leaves, and stems, acting probably as physical adhesion sites of the hyphae. D. caulivora colonized the stem rapidly reaching the phloem and the xylem at 72 h post-inoculation (hpi), and after 96 hpi, the stem was heavily colonized. Infected soybean plants induce reinforcement of the cell walls, evidenced by incorporation of phenolic compounds. In addition, several defense genes were induced in D. caulivora-inoculated stems, including those encoding a pathogenesis-related protein-1 (PR-1), a PR-10, a ß-1,3-glucanase, two chitinases, two lipoxygenases, a basic peroxidase, a defensin, a phenylalanine-ammonia lyase, and a chalcone synthase. This study provides new insights into the interaction of soybean with D. caulivora, an important pathogen causing SSC, and provides information on the activation of plant defense responses.

Genome-wide analysis of the soybean CRK-family and transcriptional regulation by biotic stress signals triggering plant immunity.

Cysteine-rich receptor-like kinases (CRKs) are transmembrane proteins that exhibit ectodomains containing the domain of unknown function 26 (DUF26). The CRKs form a large subfamily of receptor-like kinases in plants, and their possible functions remain to be elucidated. Several lines of evidence suggest that CRKs play important roles in plant defense responses to environmental stress, including plant immunity. We performed a genome-wide analysis of CRK encoding genes in soybean (Glycine max). We found 91 GmCRKs distributed in 16 chromosomes, and identified several tandem and segmental duplications, which influenced the expansion of this gene family. According to our phylogenetic analysis, GmCRKs are grouped in four clades. Furthermore, 12% of the members exhibited GmCRKs with a duplicated bi-modular organization of the ectodomains, containing four DUF26 domains. Expression analysis of GmCRKs was performed by exploring publicly available databases, and by RT-qPCR analysis of selected genes in soybean leaves responding to biotic stress signals. GmCRKs exhibited diverse expression patterns in leaves, stems, roots, and other tissues. Some of them were highly expressed in only one type of tissue, suggesting predominant roles in specific tissues. Furthermore, several GmCRKs were induced with PAMPs, DAMPs and the pathogens Phakopsora pachyrhizi and Phytophthora sojae. Expression profiles of several GmCRKs encoding highly similar proteins exhibited antagonist modes of regulation. The results suggest a fine-tuning control of GmCRKs transcriptional regulation in response to external stimuli, including PAMPs and DAMPs. This study offers a comprehensive view of the GmCRKs family in soybean, and provides a foundation for evolutionary and functional analysis of this family of plant proteins involved in the perception of pathogens and activation of plant immunity.

Adaptation Mechanisms in the Evolution of Moss Defenses to Microbes.

Bryophytes, including mosses, liverworts and hornworts are early land plants that have evolved key adaptation mechanisms to cope with abiotic stresses and microorganisms. Microbial symbioses facilitated plant colonization of land by enhancing nutrient uptake leading to improved plant growth and fitness. In addition, early land plants acquired novel defense mechanisms to protect plant tissues from pre-existing microbial pathogens. Due to its evolutionary stage linking unicellular green algae to vascular plants, the non-vascular moss Physcomitrella patens is an interesting organism to explore the adaptation mechanisms developed in the evolution of plant defenses to microbes. Cellular and biochemical approaches, gene expression profiles, and functional analysis of genes by targeted gene disruption have revealed that several defense mechanisms against microbial pathogens are conserved between mosses and flowering plants. P. patens perceives pathogen associated molecular patterns by plasma membrane receptor(s) and transduces the signal through a MAP kinase (MAPK) cascade leading to the activation of cell wall associated defenses and expression of genes that encode proteins with different roles in plant resistance. After pathogen assault, P. patens also activates the production of ROS, induces a HR-like reaction and increases levels of some hormones. Furthermore, alternative metabolic pathways are present in P. patens leading to the production of a distinct metabolic scenario than flowering plants that could contribute to defense. P. patens has acquired genes by horizontal transfer from prokaryotes and fungi, and some of them could represent adaptive benefits for resistance to biotic stress. In this review, the current knowledge related to the evolution of plant defense responses against pathogens will be discussed, focusing on the latest advances made in the model plant P. patens.

Activation of Shikimate, Phenylpropanoid, Oxylipins, and Auxin Pathways in Pectobacterium carotovorum Elicitors-Treated Moss.

Plants have developed complex defense mechanisms to cope with microbial pathogens. Pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) are perceived by pattern recognition receptors (PRRs), leading to the activation of defense. While substantial progress has been made in understanding the activation of plant defense by PAMPs and DAMPs recognition in tracheophytes, far less information exists on related processes in early divergent plants like mosses. The aim of this study was to identify genes that were induced in P. patens in response to elicitors of Pectobacterium carotovorum subsp. carotovorum, using a cDNA suppression subtractive hybridization (SSH) method. A total of 239 unigenes were identified, including genes involved in defense responses related to the shikimate, phenylpropanoid, and oxylipin pathways. The expression levels of selected genes related to these pathways were analyzed using quantitative RT-PCR, confirming their rapid induction by P.c. carotovorum derived elicitors. In addition, P. patens induced cell wall reinforcement after elicitor treatment by incorporation of phenolic compounds, callose deposition, and elevated expression of Dirigent-like encoding genes. Small molecule defense markers and phytohormones such as cinnamic acid, 12-oxo-phytodienoic acid, and auxin levels all increased in elicitor-treated moss tissues. In contrast, salicylic acid levels decreased while abscisic acid levels remained unchanged. P. patens reporter lines harboring an auxin-inducible promoter fused to ß-glucuronidase revealed GUS activity in protonemal and gametophores tissues treated with elicitors of P.c. carotovorum, consistent with a localized activation of auxin signaling. These results indicate that P. patens activates the shikimate, phenylpropanoid, oxylipins, and auxin pathways upon treatment with P.c. carotovorum derived elicitors.

Physcomitrella patens Activates Defense Responses against the Pathogen Colletotrichum gloeosporioides.

The moss Physcomitrella patens is a suitable model plant to analyze the activation of defense mechanisms after pathogen assault. In this study, we show that Colletotrichum gloeosporioides isolated from symptomatic citrus fruit infects P. patens and cause disease symptoms evidenced by browning and maceration of tissues. After C. gloeosporioides infection, P. patens reinforces the cell wall by the incorporation of phenolic compounds and induces the expression of a Dirigent-protein-like encoding gene that could lead to the formation of lignin-like polymers. C. gloeosporioides-inoculated protonemal cells show cytoplasmic collapse, browning of chloroplasts and modifications of the cell wall. Chloroplasts relocate in cells of infected tissues toward the initially infected C. gloeosporioides cells. P. patens also induces the expression of the defense genes PAL and CHS after fungal colonization. P. patens reporter lines harboring the auxin-inducible promoter from soybean (GmGH3) fused to ß-glucuronidase revealed an auxin response in protonemal tissues, cauloids and leaves of C. gloeosporioides-infected moss tissues, indicating the activation of auxin signaling. Thus, P. patens is an interesting plant to gain insight into defense mechanisms that have evolved in primitive land plants to cope with microbial pathogens.

Activation of Defense Mechanisms against Pathogens in Mosses and Flowering Plants.

During evolution, plants have developed mechanisms to cope with and adapt to different types of stress, including microbial infection. Once the stress is sensed, signaling pathways are activated, leading to the induced expression of genes with different roles in defense. Mosses (Bryophytes) are non-vascular plants that diverged from flowering plants more than 450 million years ago, allowing comparative studies of the evolution of defense-related genes and defensive metabolites produced after microbial infection. The ancestral position among land plants, the sequenced genome and the feasibility of generating targeted knock-out mutants by homologous recombination has made the moss Physcomitrella patens an attractive model to perform functional studies of plant genes involved in stress responses. This paper reviews the current knowledge of inducible defense mechanisms in P. patens and compares them to those activated in flowering plants after pathogen assault, including the reinforcement of the cell wall, ROS production, programmed cell death, activation of defense genes and synthesis of secondary metabolites and defense hormones. The knowledge generated in P. patens together with comparative studies in flowering plants will help to identify key components in plant defense responses and to design novel strategies to enhance resistance to biotic stress.

Physcomitrella patens activates reinforcement of the cell wall, programmed cell death and accumulation of evolutionary conserved defence signals, such as salicylic acid and 12-oxo-phytodienoic acid, but not jasmonic acid, upon Botrytis cinerea infection.

The moss Physcomitrella patens is an evolutionarily basal model system suitable for the analysis of plant defence responses activated after pathogen assault. Upon infection with the necrotroph Botrytis cinerea, several defence mechanisms are induced in P. patens, including the fortification of the plant cell wall by the incorporation of phenolic compounds and the induced expression of related genes. Botrytis cinerea infection also activates the accumulation of reactive oxygen species and cell death with hallmarks of programmed cell death in moss tissues. Salicylic acid (SA) levels also increase after fungal infection, and treatment with SA enhances transcript accumulation of the defence gene phenylalanine ammonia-lyase (PAL) in P. patens colonies. The expression levels of the genes involved in 12-oxo-phytodienoic acid (OPDA) synthesis, including lipoxygenase (LOX) and allene oxide synthase (AOS), increase in P. patens gametophytes after pathogen assault, together with a rise in free linolenic acid and OPDA concentrations. However, jasmonic acid (JA) could not be detected in healthy or infected tissues of this plant. Our results suggest that, although conserved defence signals, such as SA and OPDA, are synthesized and are probably involved in the defence response of P. patens against B. cinerea infection, JA production appears to be missing. Interestingly, P. patens responds to OPDA and methyl jasmonate by reducing moss colony growth and rhizoid length, suggesting that jasmonate perception is present in mosses. Thus, P. patens can provide clues with regard to the evolution of different defence pathways in plants, including signalling and perception of OPDA and jasmonates in nonflowering and flowering plants.

Multiple defence signals induced by Erwinia carotovora ssp. carotovora elicitors in potato.

SUMMARY Signal pathways involved in Solanum tuberosum-Erwinia carotovora ssp. carotovora(SCC3193) interaction were characterized. To this end, the concentration of several signal molecules implicated in plant defence such as ethylene (ET), jasmonates (JA) and salicylic acid (SA) were measured in potato plants treated by cell-free culture filtrates (CF) from E. c. carotovora(SCC3193). Furthermore, the presence of other potential signalling compounds such as cinnamic acid (CA) and related aromatic compounds was screened in the elicitor-treated plants. The activity of these signal compounds as inducers of defence-related genes such as drd-1 (a defence-related alcohol dehydrogenase), pinII (proteinase inhibitor II), chtB4 (basic chitinase) and chtA2 (acidic chitinase) was characterized. The results demonstrate that ET, JA and CA accumulate in potato tissues in response to CF. These signal molecules were shown to induce differential expression of drd-1, pinII, chtB4 and chtA2. Our data suggest that in addition to ET and JA, CA and possibly other aromatic compounds also may play a role in defence signalling in potato.

Down-regulation of photosystem I by Erwinia carotovora-derived elicitors correlates with H(2)O(2) accumulation in chloroplasts of potato.

SUMMARY Identification of Solanum tuberosum genes responsive to culture filtrates (CF) from Erwinia carotovora ssp. carotovora resulted in isolation of psaD, a nuclear gene encoding the PSI-D subunit of photosystem I (PSI). This gene was rapidly and markedly down-regulated in CF-treated or wounded plants. Down-regulation of psaD transcripts was also triggered by signal molecules involved in plant defence such as methyl jasmonate. The CF-induced down-regulation of psaD transcripts was correlated with an accumulation of hydrogen peroxide in chloroplasts and a down-regulation of the NADP(+) photoreduction activity mediated by PSI. These results suggest that the CF-induced down-regulation of PSI may be related to the accumulation of reactive oxygen species in chloroplasts of plant cells responding to E. c. carotovora.

A novel potato defence-related alcohol:NADP+ oxidoreductase induced in response to Erwinia carotovora.

Identification of Solanum tuberosum genes responsive to culture filtrates (CF) from Erwinia carotovora subsp. carotovora led to the isolation of a full-length cDNA with high sequence similarity to several alcohol dehydrogenases. Accumulation of transcripts corresponding to this defence-related alcohol dehydrogenase (drd-1) was rapidly induced in CF-treated and wounded plants. The gene was also responsive to molecules involved in defence signalling such as salicylic acid, methyl jasmonate and ethylene. To elucidate the biochemical function of DRD-1, its cDNA was expressed in Escherichia coli. Enzymatic assays revealed that DRD-1 is an alcohol:NADP+ oxidoreductase with preference for various aromatic and aliphatic aldehydes. The enzyme exhibited high activity with several aldehydes including 2-methoxybenzaldehyde, 3-methoxybenzaldehyde, salicylaldehyde, o-vanillin, cinnamaldehyde, hydrocinnamaldehyde, hexanal and octanal. Identification of the reaction product by thin-layer chromatography confirmed the reduction of aldehydes to alcohols. Enzymatic activity measured with 2-methoxybenzaldehyde as a substrate was increased in salicylic acid- or methyl jasmonate-treated plants. These data suggest that DRD-1 may play an important role in potato defence response to Erwinia carotovora.

Pathogen derived elicitors: searching for receptors in plants.

SUMMARY Recognition of potential pathogens is central to plants' ability to defend themselves against harmful microbes. Plants are able to recognize pathogen-derived molecules; elicitors that trigger a number of induced defences in plants. Microbial elicitors constitute a bewildering array of compounds including different oligosaccharides, lipids, peptides and proteins. Identifying the receptors for this vast array of elicitors is a major research challenge. Only in a very few cases has the cognate receptor for a particular elicitor been identified. Biochemical studies have resulted in the characterization of some elicitor binding proteins that may be part of the recognition complex. Transmembrane receptor-like protein kinases (RLKs) constitute one of the most likely categories of receptors involved in pathogen perception. Some of these serine/threonine kinases have been identified as resistance or R genes, others as induced by pathogens or elicitors. One of the RLKs belonging to a leucine rich repeat (LRR) class of putative receptor kinases was recently identified as a receptor for bacterial flagellin, and the underlying signal pathway leading to activation of defence genes was elucidated. These and other recent studies have revealed intriguing similarities in elicitor recognition and defence signalling processes in plant and animal hosts suggesting a common evolutionary origin of eukaryotic defence mechanisms.