Showing papers in "Molecular Plant-microbe Interactions in 2015"

The Plant Microbiome at Work

Abstract: Plants host distinct microbial communities on and inside their tissues designated the plant microbiota. Microbial community profiling enabled the description of the phylogenetic structure of the plant microbiota to an unprecedented depth, whereas functional insights are largely derived from experiments using individual microorganisms. The binary interplay between isolated members of the plant microbiota and host plants ranges from mutualistic to commensalistic and pathogenic relationships. However, how entire microbial communities capable of executing both growth-promoting and growth-compromising activities interfere with plant fitness remains largely unknown. Ultimately, unravelling the net result of microbial activities encoded in the extended plant genome-the plant microbiome-will be key to understanding and exploiting the full yield potential of a crop plant. In this perspective, we summarize first achievements of plant-microbiome research, we discuss future research directions, and we provide ideas for the translation of basic science to application to capitalize on the plant microbiome at work.

Seasonal community succession of the phyllosphere microbiome

Abstract: The leaf microbiome is influenced by both biotic and abiotic factors. Currently, we know little about the relative importance of these factors in determining microbiota composition and dynamics. To explore this issue, we collected weekly leaf samples over a 98-day growing season from multiple cultivars of common bean, soybean, and canola planted at three locations in Ontario, Canada, and performed Illumina-based microbiome analysis. We find that the leaf microbiota at the beginning of the season is very strongly influenced by the soil microbiota but, as the season progresses, it differentiates, becomes significantly less diverse, and transitions to having a greater proportion of leaf-specific taxa that are shared among all samples. A phylogenetic investigation of communities by reconstruction of unobserved states imputation of microbiome function inferred from the taxonomic data found significant differences between the soil and leaf microbiome, with a significant enrichment of motility gene categories in the former and metabolic gene categories in the latter. A network co-occurrence analysis identified two highly connected clusters as well as subclusters of putative pathogens and growth-promoting bacteria. These data reveal some of the complex ecological dynamics that occur in microbial communities over the course of a growing season and highlight the importance of community succession.

Linking Jasmonic Acid Signaling, Root Exudates, and Rhizosphere Microbiomes.

Abstract: Jasmonic acid (JA) is an essential hormone in plant development and defense responses in Arabidopsis thaliana. Exogenous treatment with JA has recently been shown to alter root exudate profiles and the composition of root-associated bacterial communities. However, it is currently unknown whether disruptions of the JA in the rhizosphere affect root exudation profiles and the relative abundance of bacteria and archaea in the rhizosphere. In the present study, two Arabidopsis mutants that are disrupted in different branches of the jasmonate pathway, namely myc2 and med25, were cultivated in nutrient solution and soil to profile root exudates and bacterial and archaeal communities, respectively. Compared with the wild type, both mutants showed distinct exudation patterns, including lower amounts of asparagine, ornithine, and tryptophan, as well as distinct bacterial and archaeal community composition, as illustrated by an increased abundance of Streptomyces, Bacillus, and Lysinibacillus taxa in the med25 rhizosphere and an Enterobacteriaceae population in myc2. Alternatively, the Clostridiales population was less abundant in the rhizosphere of both mutants. Similarities between plant genotypes were highly correlated, as determined by operational taxonomic units in the rhizosphere and metabolites in root exudates. This strongly suggests that root exudates play a major role in modulating changes in microbial community composition upon plant defense responses.

Genome, Transcriptome, and Functional Analyses of Penicillium expansum Provide New Insights Into Secondary Metabolism and Pathogenicity

Abstract: The relationship between secondary metabolism and infection in pathogenic fungi has remained largely elusive. The genus Penicillium comprises a group of plant pathogens with varying host specificities and with the ability to produce a wide array of secondary metabolites. The genomes of three Penicillium expansum strains, the main postharvest pathogen of pome fruit, and one Pencillium italicum strain, a postharvest pathogen of citrus fruit, were sequenced and compared with 24 other fungal species. A genomic analysis of gene clusters responsible for the production of secondary metabolites was performed. Putative virulence factors in P. expansum were identified by means of a transcriptomic analysis of apple fruits during the course of infection. Despite a major genome contraction, P. expansum is the Penicillium species with the largest potential for the production of secondary metabolites. Results using knockout mutants clearly demonstrated that neither patulin nor citrinin are required by P. expansum to successfully infect apples. Li et al. ( MPMI-12-14-0398-FI ) reported similar results and conclusions in their recently accepted paper.

Cyclic Lipopeptides of Bacillus amyloliquefaciens subsp. plantarum Colonizing the Lettuce Rhizosphere Enhance Plant Defense Responses Toward the Bottom Rot Pathogen Rhizoctonia solani

Abstract: The commercially available inoculant Bacillus amyloliquefaciens FZB42 is able to considerably reduce lettuce bottom rot caused by Rhizoctonia solani. To understand the interaction between FZB42 and R. solani in the rhizosphere of lettuce, we used an axenic system with lettuce bacterized with FZB42 and inoculated with R. solani. Confocal laser scanning microscopy showed that FZB42 could delay the initial establishment of R. solani on the plants. To show which secondary metabolites of FZB42 are produced under these in-situ conditions, we developed an ultra-high performance liquid chromatography coupled to time of flight mass spectrometry–based method and identified surfactin, fengycin, and bacillomycin D in the lettuce rhizosphere. We hypothesized that lipopeptides and polyketides play a role in enhancing the plant defense responses in addition to the direct antagonistic effect toward R. solani and used a quantitative real-time polymerase chain reaction–based assay for marker genes involved in defense signaling pathways in lettuce. A significant higher expression of PDF 1.2 observed in the bacterized plants in response to subsequent pathogen challenge showed that FZB42 could enhance the lettuce defense response toward the fungal pathogen. To identify if surfactin or other nonribosomally synthesized secondary metabolites could elicit the observed enhanced defense gene expression, we examined two mutants of FZB42 deficient in production of surfactin and the lipopetides and polyketides, by expression analysis and pot experiments. In the absence of surfactin and other nonribosomally synthesized secondary metabolites, there was no enhanced PDF 1.2–mediated response to the pathogen challenge. Pot experiment results showed that the mutants failed to reduce disease incidence in lettuce as compared with the FZB42 wild type, indicating, that surfactin as well as other nonribosomally synthesized secondary metabolites play a role in the actual disease suppression and on lettuce health. In conclusion, our study showed that nonribosomally synthesized secondary metabolites of FZB42 are actually produced in the lettuce rhizosphere and contribute to the disease suppression by mediating plant defense gene expression toward the pathogen R. solani.

Candidate Effector Proteins of the Rust Pathogen Melampsora larici-populina Target Diverse Plant Cell Compartments

Abstract: Rust fungi are devastating crop pathogens that deliver effector proteins into infected tissues to modulate plant functions and promote parasitic growth. The genome of the poplar leaf rust fungus Melampsora larici-populina revealed a large catalog of secreted proteins, some of which have been considered candidate effectors. Unraveling how these proteins function in host cells is a key to understanding pathogenicity mechanisms and developing resistant plants. In this study, we used an effectoromics pipeline to select, clone, and express 20 candidate effectors in Nicotiana benthamiana leaf cells to determine their subcellular localization and identify the plant proteins they interact with. Confocal microscopy revealed that six candidate effectors target the nucleus, nucleoli, chloroplasts, mitochondria, and discrete cellular bodies. We also used coimmunoprecipitation (coIP) and mass spectrometry to identify 606 N. benthamiana proteins that associate with the candidate effectors. Five candidate effectors specifically associated with a small set of plant proteins that may represent biologically relevant interactors. We confirmed the interaction between the candidate effector MLP124017 and TOPLESS-related protein 4 from poplar by in planta coIP. Altogether, our data enable us to validate effector proteins from M. larici-populina and reveal that these proteins may target multiple compartments and processes in plant cells. It also shows that N. benthamiana can be a powerful heterologous system to study effectors of obligate biotrophic pathogens.

Genomic Characterization Reveals Insights Into Patulin Biosynthesis and Pathogenicity in Penicillium Species

Abstract: Penicillium species are fungal pathogens that infect crop plants worldwide. P. expansum differs from P. italicum and P. digitatum, all major postharvest pathogens of pome and citrus, in that the fo...

Synergistic Action of a Metalloprotease and a Serine Protease from Fusarium oxysporum f. sp. lycopersici Cleaves Chitin-Binding Tomato Chitinases, Reduces Their Antifungal Activity, and Enhances Fungal Virulence

Abstract: As part of their defense strategy against fungal pathogens, plants secrete chitinases that degrade chitin, the major structural component of fungal cell walls. Some fungi are not sensitive to plant chitinases because they secrete chitin-binding effector proteins that protect their cell wall against these enzymes. However, it is not known how fungal pathogens that lack chitin-binding effectors overcome this plant defense barrier. Here, we investigated the ability of fungal tomato pathogens to cleave chitin-binding domain (CBD)-containing chitinases and its effect on fungal virulence. Four tomato CBD chitinases were produced in Pichia pastoris and were incubated with secreted proteins isolated from seven fungal tomato pathogens. Of these, Fusarium oxysporum f. sp. lycopersici, Verticillium dahliae, and Botrytis cinerea were able to cleave the extracellular tomato chitinases SlChi1 and SlChi13. Cleavage by F. oxysporum removed the CBD from the N-terminus, shown by mass spectrometry, and significantly reduced the chitinase and antifungal activity of both chitinases. Both secreted metalloprotease FoMep1 and serine protease FoSep1 were responsible for this cleavage. Double deletion mutants of FoMep1 and FoSep1 of F. oxysporum lacked chitinase cleavage activity on SlChi1 and SlChi13 and showed reduced virulence on tomato. These results demonstrate the importance of plant chitinase cleavage in fungal virulence.

Recent Progress in RXLR Effector Research.

Abstract: Some of the most devastating oomycete pathogens deploy effector proteins, with the signature amino acid motif RXLR, that enter plant cells to promote virulence. Research on the function and evolution of RXLR effectors has been very active over the decade that has transpired since their discovery. Comparative genomics indicate that RXLR genes play a major role in virulence for Phytophthora and downy mildew species. Importantly, gene-for-gene resistance against these oomycete lineages is based on recognition of RXLR proteins. Comparative genomics have revealed several mechanisms through which this resistance can be broken, most notably involving epigenetic control of RXLR gene expression. Structural studies have revealed a core fold that is present in the majority of RXLR proteins, providing a foundation for detailed mechanistic understanding of virulence and avirulence functions. Finally, functional studies have demonstrated that suppression of host immunity is a major function for RXLR proteins. Host protein targets are being identified in a variety of plant cell compartments. Some targets comprise hubs that are also manipulated by bacteria and fungi, thereby revealing key points of vulnerability in the plant immune network.

Pi64, Encoding a Novel CC-NBS-LRR Protein, Confers Resistance to Leaf and Neck Blast in Rice

Abstract: Rice blast caused by Magnaporthe oryzae poses a major threat to rice production worldwide. The utilization of host resistance (R) genes is considered to be the most effective and economic means to control rice blast. Here, we show that the japonica landrace Yangmaogu (YMG) displays a broader spectrum of resistance to blast isolates than other previously reported broad-spectrum resistant (BSR) cultivars. Genetic analysis suggested that YMG contains at least three major R genes. One gene, Pi64, which exhibits resistance to indica-sourced isolate CH43 and several other isolates, was mapped to a 43-kb interval on chromosome 1 of YMG. Two open reading frames (NBS-1 and NBS-2) encoding nucleotide-binding site and leucine-rich repeat proteins were short-listed as candidate genes for Pi64. Constructs containing each candidate gene were transformed into three susceptible japonica cultivars. Only transformants with NBS-2 conferred resistance to leaf and neck blast, validating the idea that NBS-2 represents the functional Pi64 gene. Pi64 is constitutively expressed at all development stages and in all tissues examined. Pi64 protein is localized in both the cytoplasm and nucleus. Furthermore, introgression of Pi64 into susceptible cultivars via gene transformation and marker-assisted selection conferred high-level and broad-spectrum leaf and neck blast resistance to indica-sourced isolates, demonstrating its potential utility in breeding BSR rice cultivars.

Psychrotolerant Endophytic Pseudomonas sp. Strains OB155 and OS261 Induced Chilling Resistance in Tomato Plants (Solanum lycopersicum Mill.) by Activation of Their Antioxidant Capacity.

Abstract: Studies on chilling stress damage and its mitigation through microorganisms in members of family Solanaceae is limited, despite their economic importance. We studied chilling stress alleviation in tomato plants colonized by psychrotolerant bacterial strains Pseudomonas vancouverensis OB155-gfp and P. frederiksbergensis OS261-gfp. Log phase cultures of bacterial strains were coated on surface-sterilized seeds (bacterization) before sowing and nonbacterized (control) seeds were coated with sterile bacterial growth medium. All plants were grown at temperatures of 30 and 25°C and at the end of 4 weeks, chilling treatment (12 and 10°C) was imposed for 1 week on half of the bacterized and control plants. Under normal conditions (30 and 25°C), no significant difference was observed in antioxidant activity, proline accumulation, and expression of cold acclimation genes in tomato leaf tissues of both control and bacterized plants. However, plants exposed to temperatures of 12 and 10°C were found to decrease in robustness and nutrient uptake, accompanied by increased membrane damage. Chilling resistance in bacterized plants was evident from reduced membrane damage and reactive oxygen species levels, improved antioxidant activity in leaf tissues, and high expression of cold acclimation genes LeCBF1 and LeCBF3 compared with control plants. Confocal microscopy confirmed effective colonization and intercellular localization of cold-adapted bacterial strains OB155-gfp and OS261-gfp.

Plant-Influenced Gene Expression in the Rice Endophyte Burkholderia kururiensis M130

Abstract: Burkholderia kururiensis M130 is one of the few rice endophytic diazotrophic bacteria identified thus far which is able to enhance growth of rice. To date, very little is known of how strain M130 and other endophytes enter and colonize plants. Here, we identified genes of strain M130 that are differentially regulated in the presence of rice plant extract. A genetic screening of a promoter probe transposon mutant genome bank and RNAseq analysis were performed. The screening of 10,100 insertions of the genomic transposon reporter library resulted in the isolation of 61 insertions displaying differential expression in response to rice macerate. The RNAseq results validated this screen and indicated that this endophytic bacterium undergoes major changes in the presence of plant extract regulating 27.7% of its open reading frames. A large number of differentially expressed genes encode membrane transporters and secretion systems, indicating that the exchange of molecules is an important aspect of bacterial endophytic growth. Genes related to motility, chemotaxis, and adhesion were also overrepresented, further suggesting plant–bacteria interaction. This work highlights the potential close signaling taking place between plants and bacteria and helps us to begin to understand the adaptation of an endophyte in planta.

Transgenic Wheat Expressing a Barley UDP-Glucosyltransferase Detoxifies Deoxynivalenol and Provides High Levels of Resistance to Fusarium graminearum

Abstract: Fusarium head blight (FHB), mainly caused by Fusarium graminearum, is a devastating disease of wheat that results in economic losses worldwide. During infection, F. graminearum produces trichothecene mycotoxins, including deoxynivalenol (DON), that increase fungal virulence and reduce grain quality. Transgenic wheat expressing a barley UDP-glucosyltransferase (HvUGT13248) were developed and evaluated for FHB resistance, DON accumulation, and the ability to metabolize DON to the less toxic DON-3-O-glucoside (D3G). Point-inoculation tests in the greenhouse showed that transgenic wheat carrying HvUGT13248 exhibited significantly higher resistance to disease spread in the spike (type II resistance) compared with nontransformed controls. Two transgenic events displayed complete suppression of disease spread in the spikes. Expression of HvUGT13248 in transgenic wheat rapidly and efficiently conjugated DON to D3G, suggesting that the enzymatic rate of DON detoxification translates to type II resistance. Under field conditions, FHB severity was variable; nonetheless, transgenic events showed significantly less-severe disease phenotypes compared with the nontransformed controls. In addition, a seedling assay demonstrated that the transformed plants had a higher tolerance to DON-inhibited root growth than nontransformed plants. These results demonstrate the utility of detoxifying DON as a FHB control strategy in wheat.

Mitogen-Activated Protein Kinase 6 and Ethylene and Auxin Signaling Pathways Are Involved in Arabidopsis Root-System Architecture Alterations by Trichoderma atroviride

Abstract: Trichoderma atroviride is a symbiotic fungus that interacts with roots and stimulates plant growth and defense. Here, we show that Arabidopsis seedlings cocultivated with T. atroviride have an altered root architecture and greater biomass compared with axenically grown seedlings. These effects correlate with increased activity of mitogen-activated protein kinase 6 (MPK6). The primary roots of mpk6 mutants showed an enhanced growth inhibition by T. atroviride when compared with wild-type (WT) plants, while T. atroviride increases MPK6 activity in WT roots. It was also found that T. atroviride produces ethylene (ET), which increases with l-methionine supply to the fungal growth medium. Analysis of growth and development of WT seedlings and etr1, ein2, and ein3 ET-related Arabidopsis mutants indicates a role for ET in root responses to the fungus, since etr1 and ein2 mutants show defective root-hair induction and enhanced primary-root growth inhibition when cocultivated with T. atroviride. Increased MPK6 activity was evidenced in roots of ctr1 mutants, which correlated with repression of primary root growth, thus connecting MPK6 signaling with an ET response pathway. Auxin-inducible gene expression analysis using the DR5:uidA reporter construct further revealed that ET affects auxin signaling through the central regulator CTR1 and that fungal-derived compounds, such as indole-3-acetic acid and indole-3-acetaldehyde, induce MPK6 activity. Our results suggest that T. atroviride likely alters root-system architecture modulating MPK6 activity and ET and auxin action.

14-3-3 Proteins in Plant-Pathogen Interactions

Abstract: 14-3-3 proteins define a eukaryotic-specific protein family with a general role in signal transduction. Primarily, 14-3-3 proteins act as phosphosensors, binding phosphorylated client proteins and modulating their functions. Since phosphorylation regulates a plethora of different physiological responses in plants, 14-3-3 proteins play roles in multiple signaling pathways, including those controlling metabolism, hormone signaling, cell division, and responses to abiotic and biotic stimuli. Increasing evidence supports a prominent role of 14-3-3 proteins in regulating plant immunity against pathogens at various levels. In this review, potential links between 14-3-3 function and the regulation of plant-pathogen interactions are discussed, with a special focus on the regulation of 14-3-3 proteins in response to pathogen perception, interactions between 14-3-3 proteins and defense-related proteins, and 14-3-3 proteins as targets of pathogen effectors.

The Novel Lipopeptide Poaeamide of the Endophyte Pseudomonas poae RE*1-1-14 Is Involved in Pathogen Suppression and Root Colonization.

Abstract: Endophytic Pseudomonas poae strain RE*1-1-14 was originally isolated from internal root tissue of sugar beet plants and shown to suppress growth of the fungal pathogen Rhizoctonia solani both in vitro and in the field. To identify genes involved in its biocontrol activity, RE*1-1-14 random mutagenesis and sequencing led to the identification of a nonribosomal peptide synthetase (NRPS) gene cluster predicted to encode a lipopeptide (LP) with a 10-amino-acid peptide moiety. The two unlinked gene clusters consisted of three NRPS genes, designated poaA (cluster 1) and poaB and poaC (cluster 2), spanning approximately 33.7 kb. In silico analysis followed by chemical analyses revealed that the encoded LP, designated poaeamide, is a structurally new member of the orfamide family. Poaeamide inhibited mycelial growth of R. solani and different oomycetes, including Phytophthora capsici, P. infestans, and Pythium ultimum. The novel LP was shown to be essential for swarming motility of strain RE*1-1-14 and had an impact on root colonization of sugar beet seedlings The poaeamide-deficient mutant colonized the rhizosphere and upper plant cortex at higher densities and with more scattered colonization patterns than the wild type. Collectively, these results indicate that Pseudomonas poae RE*1-1-14 produces a structurally new LP that is relevant for its antagonistic activity against soilborne plant pathogens and for colonization of sugar beet roots.

Multiple Roles and Effects of a Novel Trichoderma Hydrophobin

Abstract: Fungi belonging to the genus Trichoderma are among the most active and ecologically successful microbes found in natural environments, because they are able to use a variety of substrates and affec...

Tomato I2 Immune Receptor Can Be Engineered to Confer Partial Resistance to the Oomycete Phytophthora infestans in Addition to the Fungus Fusarium oxysporum

Abstract: Plants and animals rely on immune receptors, known as nucleotide-binding domain and leucine-rich repeat (NLR)-containing proteins, to defend against invading pathogens and activate immune responses. How NLR receptors respond to pathogens is inadequately understood. We previously reported single-residue mutations that expand the response of the potato immune receptor R3a to AVR3a(EM), a stealthy effector from the late blight oomycete pathogen Phytophthora infestans. I2, another NLR that mediates resistance to the will-causing fungus Fusarium oxysporum f. sp. lycopersici, is the tomato ortholog of R3a. We transferred previously identified R3a mutations to I2 to assess the degree to which the resulting I2 mutants have an altered response. We discovered that wild-type I2 protein responds weakly to AVR3a. One mutant in the N-terminal coiled-coil domain, I2(I141N), appeared sensitized and displayed markedly increased response to AVR3a. Remarkably, I2(I141N) conferred partial resistance to P. infestans. Further, I2(I141N) has an expanded response spectrum to F. oxysporum f. sp. lycopersici effectors compared with the wild-type I2 protein. Our results suggest that synthetic immune receptors can be engineered to confer resistance to phylogenetically divergent pathogens and indicate that knowledge gathered for one NLR could be exploited to improve NLR from other plant species.

Tomato Whole Genome Transcriptional Response to Tetranychus urticae Identifies Divergence of Spider Mite-Induced Responses Between Tomato and Arabidopsis

Abstract: The two-spotted spider mite Tetranychus urticae is one of the most significant mite pests in agriculture, feeding on more than 1,100 plant hosts, including model plants Arabidopsis thaliana and tomato, Solanum lycopersicum. Here, we describe timecourse tomato transcriptional responses to spider mite feeding and compare them with Arabidopsis in order to determine conserved and divergent defense responses to this pest. To refine the involvement of jasmonic acid (JA) in mite-induced responses and to improve tomato Gene Ontology annotations, we analyzed transcriptional changes in the tomato JA-signaling mutant defenseless1 (def-1) upon JA treatment and spider mite herbivory. Overlay of differentially expressed genes (DEG) identified in def-1 onto those from the timecourse experiment established that JA controls expression of the majority of genes differentially regulated by herbivory. Comparison of defense responses between tomato and Arabidopsis highlighted 96 orthologous genes (of 2,133 DEG) that were recruited for defense against spider mites in both species. These genes, involved in biosynthesis of JA, phenylpropanoids, flavonoids, and terpenoids, represent the conserved core of induced defenses. The remaining tomato DEG support the establishment of tomato-specific defenses, indicating profound divergence of spider mite-induced responses between tomato and Arabidopsis.

The VELVET Complex in the Gray Mold Fungus Botrytis cinerea: Impact of BcLAE1 on Differentiation, Secondary Metabolism, and Virulence.

Abstract: Botrytis cinerea, the gray mold fungus, is an important plant pathogen. Field populations are characterized by variability with regard to morphology, the mode of reproduction (conidiation or sclerotia formation), the spectrum of secondary metabolites (SM), and virulence. Natural variation in bcvel1 encoding the ortholog of Aspergillus nidulans VeA, a member of the VELVET complex, was previously shown to affect light-dependent differentiation, the formation of oxalic acid (OA), and virulence. To gain broader insight into the B. cinerea VELVET complex, an ortholog of A. nidulans LaeA, BcLAE1, a putative interaction partner of BcVEL1, was studied. BcVEL1 but not its truncated versions interacts with BcLAE1 and BcVEL2 (VelB ortholog). In accordance with the expected common as well as specific functions of BcVEL1 and BcLAE1, the deletions of both genes result in similar though not identical phenotypes. Both mutants lost the ability to produce OA, to colonize the host tissue, and to form sclerotia. However, mutants differ with regard to aerial hyphae and conidia formation. Genome-wide expression analyses revealed that BcVEL1 and BcLAE1 have common and distinct target genes. Some of the genes that are underexpressed in both mutants, e.g., those encoding SM-related enzymes, proteases, and carbohydrate-active enzymes, may account for their reduced virulence.

Identification of a 12-gene Fusaric Acid Biosynthetic Gene Cluster in Fusarium Species Through Comparative and Functional Genomics.

Abstract: In fungi, genes involved in biosynthesis of a secondary metabolite (SM) are often located adjacent to one another in the genome and are coordinately regulated. These SM biosynthetic gene clusters typically encode enzymes, one or more transcription factors, and a transport protein. Fusaric acid is a polyketide-derived SM produced by multiple species of the fungal genus Fusarium. This SM is of concern because it is toxic to animals and, therefore, is considered a mycotoxin and may contribute to plant pathogenesis. Preliminary descriptions of the fusaric acid (FA) biosynthetic gene (FUB) cluster have been reported in two Fusarium species, the maize pathogen F. verticillioides and the rice pathogen F. fujikuroi. The cluster consisted of five genes and did not include a transcription factor or transporter gene. Here, analysis of the FUB region in F. verticillioides, F. fujikuroi, and F. oxysporum, a plant pathogen with multiple hosts, indicates the FUB cluster consists of at least 12 genes (FUB1 to FUB12). Deletion analysis confirmed that nine FUB genes, including two Zn(II)2Cys6 transcription factor genes, are required for production of wild-type levels of FA. Comparisons of FUB cluster homologs across multiple Fusarium isolates and species revealed insertion of non-FUB genes at one or two locations in some homologs. Although the ability to produce FA contributed to the phytotoxicity of F. oxysporum culture extracts, lack of production did not affect virulence of F. oxysporum on cactus or F. verticillioides on maize seedlings. These findings provide new insights into the genetic and biochemical processes required for FA production.

Identification and Characterization of the SnTox6-Snn6 Interaction in the Parastagonospora nodorum-Wheat Pathosystem.

Abstract: Parastagonospora nodorum is a necrotrophic fungal pathogen that causes Septoria nodorum blotch (SNB) (formerly Stagonospora nodorum blotch) on wheat. P. nodorum produces necrotrophic effectors (NE) that are recognized by dominant host sensitivity gene products resulting in disease development. The NE–host interaction is critical to inducing NE-triggered susceptibility (NETS). To date, seven NE–host sensitivity gene interactions, following an inverse gene-for-gene model, have been identified in the P. nodorum–wheat pathosystem. Here, we used a wheat mapping population that segregated for sensitivity to two previously characterized interactions (SnTox1-Snn1 and SnTox3-Snn3-B1) to identify and characterize a new interaction involving the NE designated SnTox6 and the host sensitivity gene designated Snn6. SnTox6 is a small secreted protein that induces necrosis on wheat lines harboring Snn6. Sensitivity to SnTox6, conferred by Snn6, was light-dependent and was shown to underlie a major disease susceptibility ...

β-Aminobutyric Acid–Induced Resistance Against Root-Knot Nematodes in Rice Is Based on Increased Basal Defense

Abstract: The nonprotein amino acid β-aminobutyric acid (BABA) is known to protect plants against various pathogens. The mode of action is relatively diverse and specific in different plant-pathogen systems. To extend the analysis of the mode of action of BABA to plant-parasitic nematodes in monocot plants, we evaluated the effect of BABA against the root-knot nematode (RKN) Meloidogyne graminicola in rice. BABA treatment of rice plants inhibited nematode penetration and resulted in delayed nematode and giant cell development. BABA-induced resistance (BABA-IR) was still functional in mutants or transgenics defective in salicylic acid biosynthesis and response or abscisic acid (ABA) response. Pharmacological inhibition of jasmonic acid (JA) and ethylene (ET) biosynthesis indicated that BABA-IR against rice RKN likely occurs independent of JA and ET. However, histochemical and biochemical quantification in combination with quantitative real-time reverse transcription-polymerase chain reaction data suggest that BABA protects rice against RKN through the activation of basal defense mechanisms of the plant, such as reactive oxygen species accumulation, lignin formation, and callose deposition.

Arabidopsis Lectin Receptor Kinases LecRK-IX.1 and LecRK-IX.2 Are Functional Analogs in Regulating Phytophthora Resistance and Plant Cell Death

Abstract: L-type lectin receptor kinases (LecRK) are potential immune receptors. Here, we characterized two closely-related Arabidopsis LecRK, LecRK-IX.1 and LecRK-IX.2, of which T-DNA insertion mutants showed compromised resistance to Phytophthora brassicae and Phytophthora capsici, with double mutants showing additive susceptibility. Overexpression of LecRK-IX.1 or LecRK-IX.2 in Arabidopsis and transient expression in Nicotiana benthamiana increased Phytophthora resistance but also induced cell death. Phytophthora resistance required both the lectin domain and kinase activity, but for cell death, the lectin domain was not needed. Silencing of the two closely related mitogen-activated protein kinase genes NbSIPK and NbNTF4 in N. benthamiana completely abolished LecRK-IX.1-induced cell death but not Phytophthora resistance. Liquid chromatography-mass spectrometry analysis of protein complexes coimmunoprecipitated in planta with LecRK-IX.1 or LecRK-IX.2 as bait, resulted in the identification of the N. benthamiana ABC transporter NbPDR1 as a potential interactor of both LecRK. The closest homolog of NbPDR1 in Arabidopsis is ABCG40, and coimmunoprecipitation experiments showed that ABCG40 associates with LecRK-IX.1 and LecRK-IX.2 in planta. Similar to the LecRK mutants, ABCG40 mutants showed compromised Phytophthora resistance. This study shows that LecRK-IX.1 and LecRK-IX.2 are Phytophthora resistance components that function independent of each other and independent of the cell-death phenotype. They both interact with the same ABC transporter, suggesting that they exploit similar signal transduction pathways.

Prophage-Encoded Peroxidase in ‘Candidatus Liberibacter asiaticus’ Is a Secreted Effector That Suppresses Plant Defenses

Abstract: ‘Candidatus Liberibacter asiaticus’ is transmitted by psyllids and causes huanglongbing (HLB), a lethal disease of citrus. Most pathogenic ‘Ca. L. asiaticus’ strains carry two nearly identical prop...

Bradyrhizobium BclA Is a Peptide Transporter Required for Bacterial Differentiation in Symbiosis with Aeschynomene Legumes

Abstract: Nodules of legume plants are highly integrated symbiotic systems shaped by millions of years of evolution They harbor nitrogen-fixing rhizobium bacteria called bacteroids Several legume species produce peptides called nodule-specific cysteine-rich (NCR) peptides in the symbiotic nodule cells which house the bacteroids NCR peptides are related to antimicrobial peptides of innate immunity They induce the endosymbionts into a differentiated, enlarged, and polyploid state The bacterial symbionts, on their side, evolved functions for the response to the NCR peptides Here, we identified the bclA gene of Bradyrhizobium sp strains ORS278 and ORS285, which is required for the formation of differentiated and functional bacteroids in the nodules of the NCR peptide-producing Aeschynomene legumes The BclA ABC transporter promotes the import of NCR peptides and provides protection against the antimicrobial activity of these peptides Moreover, BclA can complement the role of the related BacA transporter of Sinorhizobium meliloti, which has a similar symbiotic function in the interaction with Medicago legumes

The Genome of the Saprophytic Fungus Verticillium tricorpus Reveals a Complex Effector Repertoire Resembling That of Its Pathogenic Relatives

Abstract: Vascular wilts caused by Verticillium spp. are destructive plant diseases affecting hundreds of hosts. Only a few Verticillium spp. are causal agents of vascular wilt diseases, of which V. dahliae is the most notorious pathogen, and several V. dahliae genomes are available. In contrast, V. tricorpus is mainly known as a saprophyte and causal agent of opportunistic infections. Based on a hybrid approach that combines second and third generation sequencing, a near-gapless V. tricorpus genome assembly was obtained. With comparative genomics, we sought to identify genomic features in V. dahliae that confer the ability to cause vascular wilt disease. Unexpectedly, both species encode similar effector repertoires and share a genomic structure with genes encoding secreted proteins clustered in genomic islands. Intriguingly, V. tricorpus contains significantly fewer repetitive elements and an extended spectrum of secreted carbohydrate- active enzymes when compared with V. dahliae. In conclusion, we highlight the ...

ALD1 Regulates Basal Immune Components and Early Inducible Defense Responses in Arabidopsis

Abstract: Robust immunity requires basal defense machinery to mediate timely responses and feedback cycles to amplify defenses against potentially spreading infections. AGD2-LIKE DEFENSE RESPONSE PROTEIN 1 (ALD1) is needed for the accumulation of the plant defense signal salicylic acid (SA) during the first hours after infection with the pathogen Pseudomonas syringae and is also upregulated by infection and SA. ALD1 is an aminotransferase with multiple substrates and products in vitro. Pipecolic acid (Pip) is an ALD1-dependent bioactive product induced by P. syringae. Here, we addressed roles of ALD1 in mediating defense amplification as well as the levels and responses of basal defense machinery. ALD1 needs immune components PAD4 and ICS1 (an SA synthesis enzyme) to confer disease resistance, possibly through a transcriptional amplification loop between them. Furthermore, ALD1 affects basal defense by controlling microbial-associated molecular pattern (MAMP) receptor levels and responsiveness. Vascular exudates from uninfected ALD1-overexpressing plants confer local immunity to the wild type and ald1 mutants yet are not enriched for Pip. We infer that, in addition to affecting Pip accumulation, ALD1 produces non-Pip metabolites that play roles in immunity. Thus, distinct metabolite signals controlled by the same enzyme affect basal and early defenses versus later defense responses, respectively.

The Mutualist Laccaria bicolor Expresses a Core Gene Regulon During the Colonization of Diverse Host Plants and a Variable Regulon to Counteract Host-Specific Defenses

Abstract: The coordinated transcriptomic responses of both mutualistic ectomycorrhizal (ECM) fungi and their hosts during the establishment of symbiosis are not well-understood. This study characterizes the transcriptomic alterations of the ECM fungus Laccaria bicolor during different colonization stages on two hosts (Populus trichocarpa and Pseudotsuga menziesii) and compares this to the transcriptomic variations of P. trichocarpa across the same time-points. A large number of L. bicolor genes (≥ 8,000) were significantly regulated at the transcriptional level in at least one stage of colonization. From our data, we identify 1,249 genes that we hypothesize is the 'core' gene regulon necessary for the mutualistic interaction between L. bicolor and its host plants. We further identify a group of 1,210 genes that are regulated in a host-specific manner. This variable regulon encodes a number of genes coding for proteases and xenobiotic efflux transporters that we hypothesize act to counter chemical-based defenses simultaneously activated at the transcriptomic level in P. trichocarpa. The transcriptional response of the host plant P. trichocarpa consisted of differential waves of gene regulation related to signaling perception and transduction, defense response, and the induction of nutrient transfer in P. trichocarpa tissues. This study, therefore, gives fresh insight into the shifting transcriptomic landscape in both the colonizing fungus and its host and the different strategies employed by both partners in orchestrating a mutualistic interaction.

Salicylic Acid Inhibits the Replication of Tomato bushy stunt virus by Directly Targeting a Host Component in the Replication Complex

Abstract: Although the plant hormone salicylic acid (SA) plays a central role in signaling resistance to viral infection, the underlying mechanisms are only partially understood. Identification and characterization of SA's direct targets have been shown to be an effective strategy for dissecting the complex SA-mediated defense signaling network. In search of additional SA targets, we previously developed two sensitive approaches that utilize SA analogs in conjunction with either a photoaffinity labeling technique or surface plasmon resonance-based technology to identify and evaluate candidate SA-binding proteins (SABPs) from Arabidopsis. Using these approaches, we have now identified several members of the Arabidopsis glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein family, including two chloroplast-localized and two cytosolic isoforms, as SABPs. Cytosolic GAPDH is a well-known glycolytic enzyme; it also is an important host factor involved in the replication of Tomato bushy stunt virus (TBSV), a single-stranded RNA virus. Using a yeast cell-free extract, an in vivo yeast replication system, and plant protoplasts, we demonstrate that SA inhibits TBSV replication. SA does so by inhibiting the binding of cytosolic GAPDH to the negative (-)RNA strand of TBSV. Thus, this study reveals a novel molecular mechanism through which SA regulates virus replication.