GDSL Esterase/Lipase GELP1 Involved in the Defense of Apple Leaves against Colletotrichum gloeosporioides Infection.

GDSL esterases/lipases are a subclass of lipolytic enzymes that play critical roles in plant growth and development, stress response, and pathogen defense. However, the GDSL esterase/lipase genes involved in the pathogen response of apple remain to be identified and characterized. Thus, in this study, we aimed to analyze the phenotypic difference between the resistant variety, Fuji, and susceptible variety, Gala, during infection with C. gloeosporioides, screen for anti-disease-associated proteins in Fuji leaves, and elucidate the underlying mechanisms. The results showed that GDSL esterase/lipase protein GELP1 contributed to C. gloeosporioides infection defense in apple. During C. gloeosporioides infection, GELP1 expression was significantly upregulated in Fuji. Fuji leaves exhibited a highly resistant phenotype compared with Gala leaves. The formation of infection hyphae of C. gloeosporioides was inhibited in Fuji. Moreover, recombinant His:GELP1 protein suppressed hyphal formation during infection in vitro. Transient expression in Nicotiana benthamiana showed that GELP1-eGFP localized to the endoplasmic reticulum and chloroplasts. GELP1 overexpression in GL-3 plants increased resistance to C. gloeosporioides. MdWRKY15 expression was upregulated in the transgenic lines. Notably, GELP1 transcript levels were elevated in GL-3 after salicylic acid treatment. These results suggest that GELP1 increases apple resistance to C. gloeosporioides by indirectly regulating salicylic acid biosynthesis.

Effector Sntf2 Interacted with Chloroplast-Related Protein Mdycf39 Promoting the Colonization of Colletotrichum gloeosporioides in Apple Leaf.

Glomerella leaf spot of apple, caused by Colletotrichumgloeosporioides, is a devastating disease that leads to severe defoliation and fruit spots. The Colletotrichum species secretes a series of effectors to manipulate the host's immune response, facilitating its colonization in plants. However, the mechanism by which the effector of C. gloeosporioides inhibits the defenses of the host remains unclear. In this study, we reported a novel effector Sntf2 of C. gloeosporioides. The transient expression of SNTF2 inhibits BAX-induced cell death in tobacco plants. Sntf2 suppresses plant defense responses by reducing callose deposition and H2O2 accumulation. SNTF2 is upregulated during infection, and its deletion reduces virulence to the plant. Sntf2 is localized to the chloroplasts and interacts with Mdycf39 (a chloroplast PSII assembly factor) in apple leaves. The Mdycf39 overexpression line increases susceptibility to C. gloeosporioides, whereas the Mdycf39 transgenic silent line does not grow normally with pale white leaves, indicating that Sntf2 disturbs plant defense responses and growth by targeting Mdycf39.

Carbamoyl Phosphate Synthase Subunit CgCPS1 Is Necessary for Virulence and to Regulate Stress Tolerance in Colletotrichum gloeosporioides.

Glomerella leaf spot (GLS) is a severe infectious disease of apple whose infective area is growing gradually and thus poses a huge economic threat to the world. Different species of Colletotrichum including Colletotrichum gloeosporioides are responsible for GLS. For efficient GLS control, it is important to understand the mechanism by which the cruciferous crops and C. gloeosporioides interact. Arginine is among one of the several types of amino acids, which plays crucial role in biochemical and physiological functions of fungi. The arginine biosynthesis pathway involved in virulence among plant pathogenic fungi is poorly understood. In this study, CgCPS1 gene encoding carbamoyl phosphate synthase involved in arginine biosynthesis has been identified and inactivated experimentally. To assess the effects of CgCPS1, we knocked out CgCPS1 in C. gloeosporioides and evaluated its effects on virulence and stress tolerance. The results showed that deletion of CgCPS1 resulted in loss of pathogenicity. The Δcgcps1 mutants showed slow growth rate, defects in appressorium formation and failed to develop lesions on apple leaves and fruits leading to loss of virulence while complementation strain (CgCPS1-C) fully restored its pathogenicity. Furthermore, mutant strains showed extreme sensitivity to high osmotic stress displaying that CgCPS1 plays a vital role in stress response. These findings suggest that CgCPS1 is major factor that mediates pathogenicity in C. gloeosporioides by encoding carbamoyl phosphate that is involved in arginine biosynthesis and conferring virulence in C. gloeosporioides.

First Report of Passion Fruit Anthracnose Caused by Colletotrichum constrictum.

Passion fruit (Passiflora edulis) is widely cultivated in tropic and subtropic regions. Because of its unique and intense flavour and high acidity, passion fruit juice concentrate is used in making delectable sauces, desserts, candy, ice cream, sherbet, or blending with other fruit juices. Anthracnose of passion fruit is favored by frequent rainfall and average temperatures above 27°C. In August 2018, anthracnose on passion fruit was observed in commercial plantings in Lincang, Yunnan, China (23.88 N, 100.08 E). Symptoms included lesions of oval to irregular shapes with brown to dark brown borders. Infection covered most of the fruit surface with pink-to-dark sporulation as reported by Tarnowski and Ploetz (2010). A conidial mass from an individual sorus observed on an infected fruit was isolated and cultured on potato dextrose agar (PDA) supplemented with 50 µg ml-1 of streptomycin. From a single microscopic field, two monospore isolates were dissected using a sterile needle, subcultured, and referred to as BXG-1 and BXG-2. Morphological characters including conidia colour, size, and shape were similar between the two isolates. Conidia were aseptate and cylindrical with apex and rounded base. Conidial length ranged from 12.3 to 16.1 µm (avg. 13.5) and width ranged from 5.5 to 6.2 µm (avg. 5.7). Morphologic data were consistent with Colletotrichum constrictum (Damm et al., 2012). To further confirm the fungal species, the ribosomal internal transcribed spacer (ITS), partial sequences of actin (ACT), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ß-tubulin 2 (TUB2) were amplified and sequenced. Primers and PCR amplification were described by Damm et al. (2012). The sequences were compared to type sequences in GenBank. The results showed the ITS (GenBank accession MW828148 and MW828149), ACT (MW855882 and MW855883), CHS-1 (MW855884 and MW855885), GAPDH (MW855886 and MW855887), and TUB2 (MW855888 and MW855889) sequences of the isolates BXG-1 and BXG-2 were 98% identical with sequence data from strain CBS:128504 of C. constrictum. A maximum likelihood tree was constructed using MEGA-X version 10.1.6 (Kumar et al., 2018) based on a combined dataset of the ITS, ACT, CHS-1, GAPDH, and TUB2 sequences of BXG-1 and BXG-2, and those of 18 Colletotrichum spp. previously deposited in GenBank (Damm et al., 2012). The phylogenetic analysis showed that BXG-1 and BXG-2 belong to the C. constrictum clade. Based on morphology and DNA sequencing, BXG-1 and BXG-2 were identified as C. constrictum. To verify pathogenicity, passion fruit were sprayed with a suspension of 1 × 105 conidia ml-1. Control fruit were sprayed with sterilized water. After inoculation, fruit were incubated in an Artificial Climate Box at 27°C and 80% RH. Necrotic symptoms appeared 8 days after inoculation and were similar to those observed on fruit form the field. The pathogen was reisolated from lesions thus fulfilling Koch's postulates. C. constrictum has been reported to cause anthracnose of citrus from Australia (Wang et al., 2021) and mango from Italy (Ismail et al., 2015). To our knowledge, this is the first report of C. constrictum causing anthracnose on passion fruit worldwide, and these data will provide useful information for developing effective control strategies.

A SET domain-containing protein involved in cell wall integrity signaling and peroxisome biogenesis is essential for appressorium formation and pathogenicity of Colletotrichum gloeosporioides.

The chromatin modulator Set5 plays important regulatory roles in both cell growth and stress responses of Saccharomyces cerevisiae. However, its function in filamentous fungi remains poorly understood. Here, we report the pathogenicity-related gene CgSET5 discovered in a T-DNA insertional mutant M285 of Colletotrichum gloeosporioides. Bioinformatic analysis revealed that CgSET5 encodes a SET domain-containing protein that is a homolog of the budding yeast S. cerevisiae Set5. CgSET5 is important for hyphae growth and conidiation and is necessary for appressorium formation and pathogenicity. CgSet5 regulates appressorium formation in a mitogen-activated protein kinase-independent manner. Inactivation of CgSET5 resulted in a significant reduction in chitin content within the cell wall, indicating CgSet5 plays a vital role in cell wall integrity. CgSet5 is involved in peroxisome biogenesis. We identified CgSet5 as the histone H4 methyltransferase, which methylates the critical H4 lysine residues 5 and 8 in C. gloeosporioides. We carried out a yeast two-hybrid screen to find CgSet5 interacting partners. We found CgSet5 putatively interacts with an inorganic pyrophosphatase named CgPpa1, which co-localized in the cytoplasm with CgSet5. Finally, CgPpa1 was found to strongly interact with CgSet5 in vivo during appressorium formation by bimolecular fluorescence complementation assays. These data corroborate a complex control function of CgSet5 acting as a core pathogenic regulator, which connects cell wall integrity and peroxisome biogenesis in C. gloeosporioides.

Comparative transcriptome analysis reveals significant differences in gene expression between appressoria and hyphae in Colletotrichum gloeosporioides.

Fruit rot caused mainly by Colletotrichum gloeosporioides is a major cause of pre- and/or post-harvest diseases, which seriously constrains production, marketing, and export of fruits. To infect the host, this fungus evolves a specialized infection structure called the appressorium. Extensive past studies have characterized many appressorium-related genes in C. gloeosporioides, separately. However, a comprehensive understanding of the genes contributing to appressorium formation is far from complete. Here, global changes in gene expression were analyzed between appressoria and hyphae using RNA-Seq. We identified 4071 genes that are up-regulated in appressorium and discovered 468 unigenes that are expressed only in appressoria, compared with the fungal hyphae. Differentially expressed genes between appressoria and hyphae were assigned to 107 KEGG pathways, including metabolic pathways, secondary metabolite biosynthesis, molecular transport and signal transduction. Fourteen putative ABC transporter genes are significantly up-regulated in appressoria, and in contrast, twenty-six down-regulated. One hundred and one transcription factor genes show more than a 2-fold up-regulation in appressoria compared to hyphae. The up-regulation of 39 secreted protein candidates is observed, suggesting they may play important roles in initial infection processes. Our data demonstrate that appressorium development of C. gloeosporioides is accompanied by significant changes in gene expression, which provides novel insights to elucidate how this fungus regulates its development, pathogenicity and immune evasion.

Structural and functional analyses of genes encoding VQ proteins in apple.

Recent studies with Arabidopsis and soybean have shown that a class of valine-glutamine (VQ) motif-containing proteins interacts with some WRKY transcription factors. However, little is known about the evolution, structures, and functions of those proteins in apple. Here, we examined their features and identified 49 apple VQ genes. Our evolutional analysis revealed that the proteins could be clustered into nine groups together with their homologues in 33 species. Historically, the main characteristics of proteins in Groups I, V, VI, VII, IX, and X were thought to have been generated before the monocot-dicot split, whereas those in Groups II, III + IV, and VIII were generated after that split. In the structural analysis, apple MdVQ proteins appeared to bind only with Group I and IIc MdWRKY proteins. Meanwhile, MdVQ1, MdVQ10, MdVQ15, and MdVQ36 interacted with multiple MdVQ proteins to form heterodimers but MdVQ15 formed a homodimer. The functional analysis indicated that overexpression of some apple MdVQs in Arabidopsis and tobacco plants effected their vegetative and reproductive growth. These results provide important information about the characteristics of apple MdVQ genes and can serve as a solid foundation for further studies about the role of WRKY-VQ interactions in regulating apple developmental and defense mechanisms.

BGDMdocker: a Docker workflow for data mining and visualization of bacterial pan-genomes and biosynthetic gene clusters.

Recently, Docker technology has received increasing attention throughout the bioinformatics community. However, its implementation has not yet been mastered by most biologists; accordingly, its application in biological research has been limited. In order to popularize this technology in the field of bioinformatics and to promote the use of publicly available bioinformatics tools, such as Dockerfiles and Images from communities, government sources, and private owners in the Docker Hub Registry and other Docker-based resources, we introduce here a complete and accurate bioinformatics workflow based on Docker. The present workflow enables analysis and visualization of pan-genomes and biosynthetic gene clusters of bacteria. This provides a new solution for bioinformatics mining of big data from various publicly available biological databases. The present step-by-step guide creates an integrative workflow through a Dockerfile to allow researchers to build their own Image and run Container easily.

ABC protein CgABCF2 is required for asexual and sexual development, appressorial formation and plant infection in Colletotrichum gloeosporioides.

ATP-binding cassette (ABC) proteins are exclusively found in both prokaryotes and eukaryotes. In this study, we have characterized a gene from Glomerella leaf spot pathogen Colletotrichum gloeosporioides that encodes an ABC protein, whose function to date remains unknown. We designated this gene as CgABCF2. Deletion of CgABCF2 showed drastic reduction both growing rate and conidial production in C. gloeosporioides. The Δcgabcf2 mutant did not form the appressoria, lost the capability to infect apple and failed to form lesions on the wounded leaves and fruits. The C. gloeosporioides native CgABCF2 fully recovered defect of the Δcgabcf2 mutant. These data indicated that CgABCF2 was required for fungal development and invasion. The transcriptions of six pectolytic enzymes genes (CgPG1, CgPG2, pnl-1, pnl-2, pelA and pelB) significantly reduced in the Δcgabcf2 mutant, indicating that deletion of CgABCF2 impaired the fungal necrotrophic growth. In addition, CgABCF2 mediated sexual development through the positive regulation of the gene MAT1-2-1 expression. These results indicated that CgABCF2 underlies the complex process governing morphogenesis, sexual and asexual reproduction, appressorial formation and pathogenicity in C. gloeosporioides.

Identification of Conidiogenesis-Associated Genes in Colletotrichum gloeosporioides by Agrobacterium tumefaciens-Mediated Transformation.

The Colletotrichum gloeosporioides is one of the most significant pathogens leading to huge economic losses. To infect plants and cause disease dissemination, the fungus elaborates to produce asexual spores called conidia, which are long-lived and highly resistant to environmental stresses. Here, we report a large-scale, systematic genome-wide screening of conidiogenesis-associated genes via conidiation assays, and high-efficiency TAIL-PCRs. Of 10,210 independent transformants tested, 59 mutants exhibited significant variation in conidial production. The T-DNA right flanking sequences of 11 conidiation-related transformants were further identified, and the obtained sequences were aligned to the genome sequence to uncover the novel loci of sporogenesis. When considering together, this study provided a large number of conidial production-variation mutants and the conidiation-related genes, which will be a valuable resource for characterizing the molecular mechanisms of conidial formation in the fungus.

[Genome-wide identification and expression analysis of the WRKY gene family in peach].

The WRKY transcription factors are one of the largest families of transcriptional regulators and play diverse regulatory roles in biotic and abiotic stresses, plant growth and development processes. In this study, the WRKY DNA-binding domain (Pfam Database number: PF03106) downloaded from Pfam protein families database was exploited to identify WRKY genes from the peach (Prunus persica 'Lovell') genome using HMMER 3.0. The obtained amino acid sequences were analyzed with DNAMAN 5.0, WebLogo 3, MEGA 5.1, MapInspect and MEME bioinformatics softwares. Totally 61 peach WRKY genes were found in the peach genome. Our phylogenetic analysis revealed that peach WRKY genes were classified into three Groups: Ⅰ, Ⅱ and Ⅲ. The WRKY N-terminal and C-terminal domains of Group Ⅰ (group I-N and group I-C) were monophyletic. The Group Ⅱ was sub-divided into five distinct clades (groupⅡ-a, Ⅱ-b, Ⅱ-c, Ⅱ-d and Ⅱ-e). Our domain analysis indicated that the WRKY regions contained a highly conserved heptapeptide stretch WRKYGQK at its N-terminus followed by a zinc-finger motif. The chromosome mapping analysis showed that peach WRKY genes were distributed with different densities over 8 chromosomes. The intron-exon structure analysis revealed that structures of the WRKY gene were highly conserved in the peach. The conserved motif analysis showed that the conserved motifs 1, 2 and 3, which specify the WRKY domain, were observed in all peach WRKY proteins, motif 5 as the unknown domain was observed in group Ⅱ-d, two WRKY domains were assigned to GroupⅠ. SqRT-PCR and qRT-PCR results indicated that 16 PpWRKY genes were expressed in roots, stems, leaves, flowers and fruits at various expression levels. Our analysis thus identified the PpWRKY gene families, and future functional studies are needed to reveal its specific roles.

Genome-wide identification and analysis of the MADS-box gene family in apple.

The MADS-box gene family is one of the most widely studied families in plants and has diverse developmental roles in flower pattern formation, gametophyte cell division and fruit differentiation. Although the genome-wide analysis of this family has been performed in some species, little is known regarding MADS-box genes in apple (Malus domestica). In this study, 146 MADS-box genes were identified in the apple genome and were phylogenetically clustered into six subgroups (MIKC(c), MIKC*, Mα, Mß, Mγ and Mδ) with the MADS-box genes from Arabidopsis and rice. The predicted apple MADS-box genes were distributed across all 17 chromosomes at different densities. Additionally, the MADS-box domain, exon length, gene structure and motif compositions of the apple MADS-box genes were analysed. Moreover, the expression of all of the apple MADS-box genes was analysed in the root, stem, leaf, flower tissues and five stages of fruit development. All of the apple MADS-box genes, with the exception of some genes in each group, were expressed in at least one of the tissues tested, which indicates that the MADS-box genes are involved in various aspects of the physiological and developmental processes of the apple. To the best of our knowledge, this report describes the first genome-wide analysis of the apple MADS-box gene family, and the results should provide valuable information for understanding the classification, cloning and putative functions of this family.

Cloning and characterization of miRNAs and their targets, including a novel miRNA-targeted NBS-LRR protein class gene in apple (Golden Delicious).

MicroRNA (miRNA) has emerged as an important regulator of gene expression in plants. 146 miRNAs were identified from apple (Malus domestica cv. Golden Delicious) by bioinformatic analysis and RNA library sequencing. From these, 135 were conserved and 11 were novel miRNAs. Target analysis predicted one of the novel miRNAs, Md-miRLn11 (Malus domestica microRNA Ln11), targeted an apple nucleotide-binding site (NBS)-leucine-rich repeat (LRR) class protein coding gene (Md-NBS). 5' RACE assay confirmed the ability of Md-miRLn11 to cleave Md-NBS at the 11-12-nt position. Analysis of the expression of Md-miRLn11 and Md-NBS during the optimum invasion period in 40 apple varieties showed that the expression of Md-NBS gene in resistant varieties is higher than in susceptible varieties, with an inverse pattern for Md-miRLn11. Seedlings from the resistant apple variety 'JiGuan' were used to carry out an Agrobacterium infiltration assay, and then inoculated with the apple leaf spot disease. The result showed a clear decline of disease resistance in JiGuan apples. In contrast, the susceptible variety 'FuJi' infiltrated with the Md-NBS gene showed a significant increase in disease resistance. Based on the above results, we propose that Md-miRLn11 regulates Md-NBS gene expression in particular under the condition of pathogen infection, and that the Md-miRLn11 targeting P-loop site may regulate many NBS-LRR protein class genes in woody plants.