High-Throughput Sequencing Reveals the Regulatory Networks of Transcriptome and Small RNAs During the Defense Against Marssonina brunnea in Poplar

MicroRNAs are implicated in the adjustment of gene expression in plant response to biotic stresses. However, the regulatory networks of transcriptome and miRNAs are still poorly understood. In the present study, we ascertained the induction of genes for small RNA biosynthesis in poplar defense to a hemibiotrophic fungus Marssonina brunnea and afterward investigated the molecular regulatory networks by performing comprehensive sequencing analysis of mRNAs and small RNAs in M. brunnea-inoculated leaves. Differentially expressed genes in M. brunnea-infected poplar are mainly involved in secondary metabolisms, phytohormone pathways, the recognition of pathogens, and MAPK pathway in the plant, with real-time quantitative PCR (qPCR) validating the mRNA-seq results. Furthermore, differentially expressed miRNAs, such as MIR167_1-6, MIR167_1-12, MIR171_2-3, MIR395-13, MIR396-3, MIR396-16, MIR398-8, and MIR477-6, were identified. Through psRobot and TargetFinder programs, MIR167-1-6, MIR395-13, MIR396-3, MIR396-16, and MIR398-8 were annotated to modulate the expression of genes implicated in transportation, signaling, and biological responses of phytohormones and activation of antioxidants for plant immunity. Besides, validated differentially expressed genes involved in lignin generation, which were phenylalanine ammonia-lyase, ferulate-5-hydroxylase, cinnamyl alcohol dehydrogenase, and peroxidase 11, were selected as targets for the identification of novel miRNAs. Correspondingly, novel miRNAs, such as Novel MIR8567, Novel MIR3228, Novel MIR5913, and Novel MIR6493, were identified using the Mireap online program, which functions in the transcriptional regulation of lignin biosynthesis for poplar anti-fungal response. The present study underlines the roles of miRNAs in the regulation of transcriptome in the anti-fungal response of poplar and provides a new idea for molecular breeding of woody plants.

Genome-Wide Characterization of Dirigent Proteins in Populus: Gene Expression Variation and Expression Pattern in Response to Marssonina brunnea and Phytohormones

Marssonina brunnea causes a major disease that limits poplar growth. Lignin and lignan play essential roles in protecting plants from various biological stresses. Dirigent (DIR) proteins are thought to control the stereoselective coupling of coniferyl alcohol in the formation of lignan and lignin. DIR family members have been well studied in several plant species, but no previous detailed genome-wide analysis has been carried out in forest trees, such as poplar. We identified 40 PtDIR genes in Populus trichocarpa and classified them into three subgroups (DIR-a, DIR-b/d, and DIR-e) based on phylogenetic analyses. These genes are distributed on 11 poplar chromosomes, and 80% of PtDIRs (32/40) are intronless. The cis-element analysis inferred that PtDIRs possess many types of biological and abiotic stress-response cis-elements. We also analyzed intra- and inter-specific collinearity, which provided deep insights into the evolutionary characteristics of the poplar DIR genes. Analyses of the protein tertiary structure and critical amino acid residues showed that PtDIR7–10 and PtDIR13–16, which belong to the DIR-a subfamily, might be involved in the regio- and stereo-selectivity of bimolecular phenoxy radical coupling in poplars. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis revealed different expression patterns for the PtDIR genes of P. trichocarpa and the PeDIR genes of ‘Nanlin 895’ in various tissues. Additionally, we analyzed responses of PeDIRs to M. brunnea and different phytohormone treatments (abscisic acid, salicylic acid, methyl jasmonate, and ethylene) in ‘Nanlin 895’. The results showed that at least 18 genes responded strongly to M. brunnea, and these PeDIRs also showed significant responses to phytohormones. These results suggest that DIR genes are involved in the poplar defense response against M. brunnea, and this study will provide fundamental insights for future research on poplar DIR genes.

Draft genome sequence of Marssonina coronaria, causal agent of apple blotch, and comparisons with the Marssonina brunnea and Marssonina rosae genomes

Marssonina coronaria Ellis & Davis is a filamentous fungus in the class Leotiomycetes that causes apple blotch, an economically important disease of apples worldwide. Here, we sequenced the whole genome of M. coronaria strain NL1. The genome contained 50.3 Mb with 589 scaffolds and 9,622 protein-coding genes. A phylogenetic analysis using multiple loci and a whole-genome alignment revealed that M. coronaria is closely related to Marssonina rosae and Marssonina brunnea. A comparison of the three genomes revealed 90 species-specific carbohydrate-active enzymes, 19 of which showed atypical distributions, and 12 species-specific secondary metabolite biosynthetic gene clusters, two of which have the potential to synthesize products analogous to PR toxin and swainsonine, respectively. We identified 796 genes encoding for small secreted proteins in Marssonina spp., many encoding for unknown hypothetical proteins. In addition, we revealed the genetic architecture of the MAT1-1 and MAT1-2 mating-type loci of M. coronaria, as well as 16 tested isolates carrying either MAT1-1 idiomorph (3) or MAT1-2 idiomorph (13). Our results showed a series of species-specific carbohydrate-active enzyme, secondary metabolite biosynthetic gene clusters and small-secreted proteins that may be involved in the adaptation of Marssonina spp. to their distinct hosts. We also confirmed that M. coronaria possesses a heterothallic mating system and has outcrossing potential in nature.

Rapid and Specific Detection of the Poplar Black Spot Disease Caused by Marssonina brunnea Using Loop-Mediated Isothermal Amplification Assay

Marssonina brunnea is the main pathogen that causes poplar black spot disease, which leads to the decrease of the photosynthetic efficiency and significantly affects the production and quality of timber. Currently, no in-field diagnostic exists for M. brunnea. Here, we described a loop-mediated isothermal amplification (LAMP) assay for the rapid and sensitive detection of M. brunnea. A set of six oligonucleotide primers was designed to recognize eight distinct sequences of the internal transcribed spacer (ITS) region of M. brunnea. The LAMP assay was optimized by the combination of high specificity, sensitivity, and rapidity for the detection of less than 10 pg/μL of target genomic DNA in 60 min per reaction at 65 °C, whereas with PCR, there was no amplification of DNA with concentration less than 1 ng/μL. Among the genomic DNA of 20 fungalisolates, only the samples containing the genomic DNA of M. brunnea changed from violet to sky blue (visible to the naked eye) by using hydroxynaphthol blue (HNB) dye. No DNA was amplified from the eight other fungus species, including two other Marssonina pathogens, three other foliar fungi pathogens of poplar, and three common foliar fungal endophytes of poplar. Moreover, the detection rates of M. brunnea from artificially and naturally infected poplar leaves were 10/16 (62.5%) and 6/16 (37.5%) using PCR, respectively, while the positive-sample ratios were both 16/16 (100%) using the LAMP assay. Overall, the ITS LAMP assay established here can be a better alternative to PCR-based techniques for the specific and sensitive detection of M. brunnea in poplar endemic areas with resource-limited settings.

Jasmonic Acid- and Ethylene-Induced Mitochondrial Alternative Oxidase Stimulates Marssonina brunnea Defense in Poplar

Mitochondrial processes are implicated in plant response to biotic stress caused by viruses, actinomyces, bacteria and pests, but their function in defense against fungal invasion remains unclear. Here, we investigated the role and regulation of mitochondrial alternative oxidase (AOX) in response to black spot disease caused by the hemibiotrophic fungus Marssonina brunnea in poplar. M. brunnea inoculation induced the transcription of the AOX1a gene in the mitochondrial electron transport chain and of jasmonic acid (JA) and ethylene (ET) biosynthetic genes, with the accumulation of these phytohormones in poplar leaf, while inhibiting the transcript amount of the mitochondrial cytochrome c oxidase gene (COX6b) and genes related to salicylic acid (SA). Enhanced AOX reduced poplar susceptibility to M. brunnea with a higher ATP/ADP ratio while the repressed AOX caused the reverse effect. Exogenous JA and 1-aminocyclopropane-1-carboxylic acid (ACC, a biosynthetic precursor of ET) inhibited the transcript amount of COX6b and consequently increased the ratio of AOX pathway to total respiration. Furthermore, the transcription of CYS C1 and CYS D1 genes catalyzing cyanide metabolism was induced, while the cysteine (CYS) substrate levels reduced upon M. brunnea inoculation; exogenous JA and ACC mimicked the effect of M. brunnea infection on cysteine. Exogenous SA enhanced, while JA and ACC reduced, poplar susceptibility to M. brunnea. Moreover, inhibiting AOX completely prohibited JA- and ET-increased tolerance to M. brunnea in poplar. These observations indicate that the JA- and ET-induced mitochondrial AOX pathway triggers defense against M. brunnea in poplar. This effect probably involves cyanide. These findings deepen our understanding of plant–pathogenic fungi interactions.

Key Genes and Genetic Interactions of Plant-Pathogen Functional Modules in Poplar Infected by Marssonina brunnea

Marssonina brunnea, the causative pathogen of Marssonina leaf spot of poplars (MLSP), devastates poplar plantations by forming black spots on leaves and defoliating trees. Although MLSP has been studied for over 30 years, the key genes that function during M. brunnea infection and their effects on plant growth are poorly understood. Here, we used multigene association studies to investigate the effects of key genes in the plant-pathogen interaction pathway, as revealed by transcriptome analysis, on photosynthesis and growth in a natural population of 435 Populus tomentosa individuals. By analyzing transcriptomic changes during three stages of infection, we detected 628 transcription factor genes among the 7,611 differentially expressed genes that might be associated with basal defense responses. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses revealed that transcriptomic changes across different stages of infection lead to the reprogramming of metabolic processes possibly related to defense activation. We identified 29,399 common single-nucleotide polymorphisms (SNPs) within 221 full-length genes in plant-pathogen interaction pathways that were significantly associated with photosynthetic and growth traits. We also detected 4,460 significant epistatic pairs associated with stomatal conductance, tree diameter, and tree height. Epistasis analysis uncovered significant interactions between 2,561 SNP-SNP pairs from different functional modules in the plant-pathogen interaction pathway, revealing possible genetic interactions. This analysis revealed many key genes that function during M. brunnea infection and their potential roles in mediating photosynthesis and plant growth, shedding light on genetic interactions between functional modules in the plant-pathogen interaction pathway.

Histopathologic characterization of the process of Marssonina brunnea infection in poplar leaves

Marssonina brunnea (Ellis & Everh.) Magnus, the causative pathogen of Marssonina leaf spot of poplars (MLSP), can lead to complete defoliation and tree death. Although MLSP has been studied for over 30 years, its precise process of infection is currently unclear. In this study, we present the process of M. brunnea infection in detail using several types of microscopy. When the conidia came into contact with the poplar leaves, they developed germ tubes to attach to the leaf surface. During the first 2 days post inoculation (dpi), infection vesicles (IV) and primary hyphae (PH) formed within host cells. The plasma membranes of the host cells penetrated by IV remained unbroken and intact organelles were visible, indicating that the IV did not kill the host cell. At 3 dpi, secondary hyphae (SH) began to appear within and outside the host cells. The ultrastructural evidence indicated that the SH could kill the host cells rapidly, producing black spots on the surfaces of the leaves. These results collectively suggested that M. brunnea is a typical hemibiotrophic fungus. In addition, M. brunnea could develop intercellular infective hyphae (IH) for expansion. Our findings also confirmed that the germ tubes, IV, and SH are crucial structures for the disease interactions and showed that the two formae speciales of M. brunnea share the same infection histopathological features. This provides important insight for further research into M. brunnea.