Complete mitochondrial genome of Melia azedarach L., reveals two conformations generated by the repeat sequence mediated recombination.

Melia azedarach is a species of enormous value of pharmaceutical industries. Although the chloroplast genome of M. azedarach has been explored, the information of mitochondrial genome (Mt genome) remains surprisingly limited. In this study, we used a hybrid assembly strategy of BGI short-reads and Nanopore long-reads to assemble the Mt genome of M. azedarach. The Mt genome of M. azedarach is characterized by two circular chromosomes with 350,142 bp and 290,387 bp in length, respectively, which encodes 35 protein-coding genes (PCGs), 23 tRNA genes, and 3 rRNA genes. A pair of direct repeats (R1 and R2) were associated with genome recombination, resulting in two conformations based on the Sanger sequencing and Oxford Nanopore sequencing. Comparative analysis identified 19 homologous fragments between Mt and chloroplast genome, with the longest fragment of 12,142 bp. The phylogenetic analysis based on PCGs were consist with the latest classification of the Angiosperm Phylogeny Group. Notably, a total of 356 potential RNA editing sites were predicted based on 35 PCGs, and the editing events lead to the formation of the stop codon in the rps10 gene and the start codons in the nad4L and atp9 genes, which were verified by PCR amplification and Sanger sequencing. Taken together, the exploration of M. azedarach gap-free Mt genome provides a new insight into the evolution research and complex mitogenome architecture.

Identification and Pathogenicity of Pestalotioid Species on Alpinia oxyphylla in Hainan Province, China.

Alpinia oxyphylla is a traditional Chinese medicinal plant with a medicinal history of more than 1700 years. Ring leaf blight (RLB) disease, caused by pestalotioid species, is an important disease of A. oxyphylla, seriously affecting the yield and quality of its fruits. The causal agent of RLB disease has not been systematically identified or characterized yet. In this study, thirty-six pestalotioid strains were isolated from the leaves and stems of A. oxyphylla that was collected from six cities of Hainan province, China. Based on the multi-locus phylogeny (ITS, tef-1α and tub2) and morphological characteristic analyses, seventeen species belonging to three genera (Neopestalotiopsis, Pestalotiopsis and Pseudopestalotiopsis) were identified, and six new species (N. baotingensis, N. oblatespora, N. olivaceous, N. oxyphylla, N. wuzhishanensis and N. yongxunensis) were described. Pathogenicity tests revealed that strains of Neopestalotiopsis species caused more severe ring leaf blight on A. oxyphylla than strains of Pestalotiopsis and Pseudopestalotiopsis under wounded inoculation conditions.

The complete mitochondrial genome of Aglaia odorata, insights into its genomic structure and RNA editing sites.

Aglaia odorata, native to Guangdong, Guangxi, and Hainan provinces in China, has long been utilized as an herbal remedy in ancient China. In this study, we assembled and annotated the complete mitochondrial genome (mitogenome) of A. odorata, which spans a total length of 537,321 bp. Conformation of the A. odorata recombination was verified through PCR experiments and Sanger sequencing. We identified and annotated 35 protein-coding genes (PCGs), 22 tRNA genes, and 3 rRNA genes within the mitogenome. Analysis of repeated elements revealed the presence of 192 SSRs, 29 pairs of tandem repeats, and 333 pairs of dispersed repeats in the A. odorata mitogenome. Additionally, we analyzed codon usage and mitochondrial plastid DNAs (MTPTs). Twelve MTPTs between the plastome and mitogenome of A. odorata were identified, with a combined length of 2,501 bp, accounting for 0.47% of the mitogenome. Furthermore, 359 high-confidence C to U RNA editing sites were predicted on PCGs, and four selected RNA editing sites were specially examined to verify the creation of start and/or stop codons. Extensive genomic rearrangement was observed between A. odorata and related mitogenomes. Phylogenetic analysis based on mitochondrial PCGs were conducted to elucidate the evolutionary relationships between A. odorata and other angiosperms.

A novel double-ribonuclease toxin-antitoxin system linked to the stress response and survival of Acidovorax citrulli.

IMPORTANCE: Bacterial fruit blotch (BFB), which is caused by the seed-borne bacterium Acidovorax citrulli, is a devastating disease affecting cucurbit crops throughout the world. Although seed fermentation and treatment with disinfectants can provide effective management of BFB, they cannot completely guarantee pathogen-free seedstock, which suggests that A. citrulli is a highly stress-resistant pathogen. Toxin-antitoxin (TA) systems are common among a diverse range of bacteria and have been reported to play a role in bacterial stress response. However, there is currently much debate about the relationship between TA systems and stress response in bacteria. The current study characterized a novel TA system (Aave_1720-Aave_1719) from A. citrulli that affects both biofilm formation and survival in response to sodium hypochlorite stress. The mechanism of neutralization differed from typical TA systems as two separate mechanisms were associated with the antitoxin, which exhibited characteristics of both type II and type V TA systems. The Aave_1720-Aave_1719 system described here also constitutes the first known report of a double-ribonuclease TA system in bacteria, which expands our understanding of the range of regulatory mechanisms utilized by bacterial TA systems, providing new insight into the survival of A. citrulli in response to stress.

Deletion of pbpC Enhances Bacterial Pathogenicity on Tomato by Affecting Biofilm Formation, Exopolysaccharides Production, and Exoenzyme Activities in Clavibacter michiganensis.

Penicillin-binding proteins (PBPs) are considered essential for bacterial peptidoglycan biosynthesis and cell wall assembly. Clavibacter michiganensis is a representative Gram-positive bacterial species that causes bacterial canker in tomato. pbpC plays a significant role in maintaining cell morphological characteristics and stress responses in C. michiganensis. The current study demonstrated that the deletion of pbpC commonly enhances bacterial pathogenicity in C. michiganensis and revealed the mechanisms through which this occurs. The expression of interrelated virulence genes, including celA, xysA, xysB, and pelA, were significantly upregulated in △pbpC mutants. Compared with those in wild-type strains, exoenzyme activities, the formation of biofilm, and the production of exopolysaccharides (EPS) were significantly increased in △pbpC mutants. It is noteworthy that EPS were responsible for the enhancement in bacterial pathogenicity, with the degree of necrotic tomato stem cankers intensifying with the injection of a gradient of EPS from C. michiganensis. These findings highlight new insights into the role of pbpC affecting bacterial pathogenicity, with an emphasis on EPS, advancing the current understanding of phytopathogenic infection strategies for Gram-positive bacteria.

Transcriptional profiling of Xanthomonas campestris pv. campestris in viable but nonculturable state.

BACKGROUND: Xanthomonas campestris pv. campestris (Xcc) is an important seed-borne plant pathogenic bacteria that can cause a serious threat to cruciferous crops. Bacteria can enter into the viable but non-culturable (VBNC) state under stress conditions, and cause potential risks to agricultural production because the VBNC bacterial cells will evade culture-based detection. However, little is known about the mechanism of VBNC. Our previous study showed that Xcc could be induced into VBNC state by copper ion (Cu2+). RESULTS: Here, RNA-seq was performed to explore the mechanism of VBNC state. The results indicated that expression profiling was changed dramatically in the different VBNC stages (0 d, 1 d, 2 d and 10 d). Moreover, metabolism related pathways were enriched according to COG, GO and KEGG analysis of differentially expressed genes (DEGs). The DEGs associated with cell motility were down-regulated, whereas pathogenicity related genes were up-regulated. This study revealed that the high expression of genes related to stress response could trigger the active cells to VBNC state, while the genes involved in transcription and translation category, as well as transport and metabolism category, were ascribed to maintaining the VBNC state. CONCLUSION: This study summarized not only the related pathways that might trigger and maintain VBNC state, but also the expression profiling of genes in different survival state of bacteria under stress. It provided a new kind of gene expression profile and new ideas for studying VBNC state mechanism in X. campestris pv. campestris.

Metabolic Promiscuity of an Orphan Small Alarmone Hydrolase Facilitates Bacterial Environmental Adaptation.

Small alarmone hydrolases (SAHs) are alarmone metabolizing enzymes found in both metazoans and bacteria. In metazoans, the SAH homolog Mesh1 is reported to function in cofactor metabolism by hydrolyzing NADPH to NADH. In bacteria, SAHs are often identified in genomes with toxic alarmone synthetases for self-resistance. Here, we characterized a bacterial orphan SAH, i.e., without a toxic alarmone synthetase, in the phytopathogen Xanthomonas campestris pv. campestris (XccSAH) and found that it metabolizes both cellular alarmones and cofactors. In vitro, XccSAH displays abilities to hydrolyze multiple nucleotides, including pppGpp, ppGpp, pGpp, pppApp, and NADPH. In vivo, X. campestris pv. campestris cells lacking sah accumulated higher levels of cellular (pp)pGpp and NADPH compared to wild-type cells upon amino acid starvation. In addition, X. campestris pv. campestris mutants lacking sah were more sensitive to killing by Pseudomonas during interbacterial competition. Interestingly, loss of sah also resulted in reduced growth in amino acid-replete medium, a condition that did not induce (pp)pGpp or pppApp accumulation. Further metabolomic characterization revealed strong depletion of NADH levels in the X. campestris pv. campestris mutant lacking sah, suggesting that NADPH/NADH regulation is an evolutionarily conserved function of both bacterial and metazoan SAHs and Mesh1. Overall, our work demonstrates a regulatory role of bacterial SAHs as tuners of stress responses and metabolism, beyond functioning as antitoxins. IMPORTANCE Small alarmone hydrolases (SAHs) comprise a widespread family of alarmone metabolizing enzymes. In metazoans, SAHs have been reported to control multiple aspects of physiology and stress resistance through alarmone and NADPH metabolisms, but their physiological functions in bacteria is mostly uncharacterized except for a few reports as antitoxins. Here, we identified an SAH functioning independently of toxins in the phytopathogen Xanthomonas campestris pv. campestris. We found that XccSAH hydrolyzed multiple alarmones and NADPH in vitro, and X. campestris pv. campestris mutants lacking sah displayed increased alarmone levels during starvation, loss of interspecies competitive fitness, growth defects, and strong reduction in NADH. Our findings reveal the importance of NADPH hydrolysis by a bacterial SAH. Our work is also the first report of significant physiological roles of bacterial SAHs beyond functioning as antitoxins and suggests that SAHs have far broader physiological roles and share similar functions across domains of life.

Class A Penicillin-Binding Protein C Is Responsible for Stress Response by Regulation of Peptidoglycan Assembly in Clavibacter michiganensis.

The cell wall peptidoglycan of bacteria is essential for their survival and shape development. The penicillin-binding proteins (PBPs) are responsible for the terminal stage of peptidoglycan assembly. It has been shown that PBPC, a member of class A high-molecular-weight PBP, played an important role in morphology maintenance and stress response in Clavibacter michiganensis. Here, we reported the stress response strategies under viable but nonculturable (VBNC) state and revealed the regulation of peptidoglycan assembly by PBPC in C. michiganensis cells. Using atomic force microscopy imaging, we found that peptidoglycan of C. michiganensis cells displayed a relatively smooth and dense surface, whereas ΔpbpC was characterized by a "ridge-and-groove" surface, which was more distinctive after Cu2+ treatment. The peptidoglycan layer of wild type cells exhibited a significant increase in thickness and slight increase in cross-linkage following Cu2+ treatment. Compared with wild type, the thickness and cross-linkage of peptidoglycan decreased during log phase in ΔpbpC cells, but the peptidoglycan cross-linkage increased significantly under Cu2+ stress, while the thickness did not change. It is noteworthy that the above changes in the peptidoglycan layer resulted in a significant increase in the accumulation of amylase and exopolysaccharide in ΔpbpC. This study elucidates the role of PBPC in Gram-positive rod-shaped plant pathogenic bacterium in response to environmental stimuli by regulating the assembling of cell wall peptidoglycan, which is significant in understanding the survival of C. michiganensis under stress and the field epidemiology of tomato bacterial canker disease. IMPORTANCE Peptidoglycan of cell walls in bacteria is a cross-linked and meshlike scaffold that provides strength to withstand the external pressure. The increased cross-linkage in peptidoglycan and altered structure in VBNC cells endowed the cell wall more resistant to adversities. Here we systematically evaluated the stress response strategies in Gram-positive rod-shaped bacterium C. michiganensis log phase cells and revealed a significant increase of peptidoglycan thickness and slight increase of cross-linkage after Cu2+ treatment. Most strikingly, knocking-out of PBPC leads to a significant increase in cross-linking of peptidoglycan in response to Cu2+ treatment. Understanding the stress resistance mechanism and survival strategy of phytopathogenic bacteria is the basis of exploring bacterial physiology and disease epidemiology.

RNA-Seq Analysis Discovers the Critical Role of Rel in ppGpp Synthesis, Pathogenicity, and the VBNC State of Clavibacter michiganensis.

The viable but nonculturable (VBNC) state is a unique survival strategy of bacteria in response to stress conditions. It was confirmed that Clavibacter michiganensis, the causal agent of bacterial canker in tomato, could be induced into the VBNC state by exposure to CuSO4 in an oligotrophic solution. RNA-sequencing analysis was used to monitor the mechanisms of the VBNC state during CuSO4 induction in C. michiganensis. The results identified that numerous genes involved in stringent response, copper resistance, and stress resistance were upregulated, and some involved in cell division were downregulated significantly. The study investigated the importance of Rel, which is an essential enzyme in the synthesis of the molecular alarmone ppGpp, via the generation of a Δrel mutant and its complementation strain. Biological characterization revealed that deficiency of rel reduced the bacterial growth, production of exopolysaccharides, and pathogenicity as well as ppGpp production. The Δrel mutant increased the sensitivity to environmental stress, exhibiting reduced growth on minimal media and a propensity to enter the VBNC state in response to CuSO4. These findings have important implications for the understanding of survival mechanism and management of C. michiganensis and other phytopathogenic bacteria.

Carrier-Free Small Molecular Self-Assembly Based on Berberine and Curcumin Incorporated in Submicron Particles for Improving Antimicrobial Activity.

Nanocarrier-based pesticide formulations have been severely restricted in agriculture practices due to their high-cost preparation process, poor loading capacity, and toxicity issues. To overcome these issues, carrier-free small molecular self-assembled submicron particles (SMPs) with an improved photoactivated antimicrobial activity based on two natural microbicides berberine hydrochloride (BBR) and curcumin (CM) are constructed by noncovalent interactions through a simple and fast preparation process (solvent exchange method) without using any adjuvant. The results show that the optimized molar ratio of BBR to CM is 2:1 at pH 5 and 25 °C in an aqueous solution for the formation of B-C SMPs. The obtained B-C SMPs exhibit excellent physicochemical properties, such as uniform morphology (407 nm), low polydispersity index (0.283), and strong ζ-potential (+24.4 mV). The antibacterial activities of B-C SMPs against Pseudomonas syringae pv. lachrymans, Clavibater michiganensis subsp. Michiganensis, and Sclerotinia sclerotiorum are 4, 2, and 1.5 times that of B + C MIX, respectively, suggesting a synergistic antimicrobial activity based on BBR and CM incorporation in the submicron particles. The genotoxicity evaluation results show that the self-assembled B-C SMPs are harmless to plant cells. Therefore, due to rational utilization of natural resources (natural microbicides, sunlight, and oxygen), carrier-free small molecular self-assembled B-C SMPs with synergistic photoactivated antimicrobial activity developed by a simple and fast preparation process would have great potential for sustainable plant disease management.

Unmarked Gene Editing in Clavibacter michiganensis Using CRISPR/Cas9 and 5-Fluorocytosine Counterselection.

Plant-pathogenic bacteria in the genus Clavibacter are important quarantine species that cause considerable economic loss worldwide. The development of effective gene editing techniques and additional selectable markers is essential to expedite gene functional analysis in this important Gram-positive genus. The current study details a highly efficient unmarked CRISPR/Cas9-mediated gene editing system in Clavibacter michiganensis, which couples the expression of cas9 and single-guide RNA with homology-directed repair templates and the negative selectable marker codA::upp within a single plasmid. Initial experiments indicated that CRISPR/Cas9-mediated transformation could be utilized for both site-directed mutagenesis, in which an A to G point mutation was introduced at the 128th nucleotide of the C. michiganensis rpsL gene to generate a streptomycin-resistant mutant, and complete gene knockout, in which the deletion of the C. michiganensis celA or katA genes resulted in transformants that lacked cellulase and catalase activity, respectively. In subsequent experiments, the introduction of the codA::upp cassette into the transformation vector facilitated the counterselection of unmarked transformants by incubation in the absence of the selective antibiotic, followed by plating on M9 agar containing 5-fluorocytosine at 100 µg/ml, in which an unmarked katA mutant lacking the transformation vector was recovered. Compared with conventional homologous recombination, the unmarked CRISPR/Cas9-mediated system was more useful and convenient because it allowed the template plasmid to be reused repeatedly to facilitate the editing of multiple genes, which constitutes a major advancement that could revolutionize research into C. michiganensis and other Clavibacter spp.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The Role of RelA and SpoT on ppGpp Production, Stress Response, Growth Regulation, and Pathogenicity in Xanthomonas campestris pv. campestris.

The alarmone ppGpp plays an important role in the survival of bacteria by triggering the stringent response when exposed to environmental stress. Although Xanthomonas campestris pv. campestris (Xcc), which causes black rot disease in crucifers, is a representative species of Gram-negative phytopathogenic bacteria, relatively little is known regarding the factors influencing the stringent response in this species. However, previous studies in other Gram-negative bacteria have indicated that RelA and SpoT play a critical role in ppGpp synthesis. The current study found that these proteins also had an important role in Xcc, with a ΔrelAΔspoT double mutant being unable to produce ppGpp, resulting in changes to phenotype including reduced production of exopolysaccharides (EPS), exoenzymes, and biofilm, as well the loss of swarming motility and pathogenicity. The ppGpp-deficient mutant also exhibited greater sensitivity to environment stress, being almost incapable of growth on modified minimal medium (mMM) and having a much greater propensity to enter the viable but nonculturable (VBNC) state in response to oligotrophic conditions (0.85% NaCl). These findings much advance our understanding of the role of ppGpp in the biology of Xcc and could have important implications for more effective management of this important pathogen. IMPORTANCE Xanthomonas campestris pv. campestris (Xcc) is a typical seedborne phytopathogenic bacterium that causes large economic losses worldwide, and this is the first original research article to investigate the role of ppGpp in this important species. Here, we revealed the function of RelA and SpoT in ppGpp production, physiology, pathogenicity, and stress resistance in Xcc. Most intriguingly, we found that ppGpp levels and downstream ppGpp-dependent phenotypes were mediated predominantly by SpoT, with RelA having only a supplementary role. Taken together, the results of the current study provide new insight into the role of ppGpp in the biology of Xcc, which could also have important implications for the role of ppGpp in the survival and pathogenicity of other pathogenic bacteria.

Polycistronic Artificial microRNA-Mediated Resistance to Cucumber Green Mottle Mosaic Virus in Cucumber.

Cucumber green mottle mosaic virus (CGMMV), as a typical seed-borne virus, causes costly and devastating diseases in the vegetable trade worldwide. Genetic sources for resistance to CGMMV in cucurbits are limited, and environmentally safe approaches for curbing the accumulation and spread of seed-transmitted viruses and cultivating completely resistant plants are needed. Here, we describe the design and application of RNA interference-based technologies, containing artificial microRNA (amiRNA) and synthetic trans-acting small interfering RNA (syn-tasiRNA), against conserved regions of different strains of the CGMMV genome. We used a rapid transient sensor system to identify effective anti-CGMMV amiRNAs. A virus seed transmission assay was developed, showing that the externally added polycistronic amiRNA and syn-tasiRNA can successfully block the accumulation of CGMMV in cucumber, but different virulent strains exhibited distinct influences on the expression of amiRNA due to the activity of the RNA-silencing suppressor. We also established stable transgenic cucumber plants expressing polycistronic amiRNA, which conferred disease resistance against CGMMV, and no sequence mutation was observed in CGMMV. This study demonstrates that RNA interference-based technologies can effectively prevent the occurrence and accumulation of CGMMV. The results provide a basis to establish and fine-tune approaches to prevent and treat seed-based transmission viral infections.

Genome Sequence Analysis of the Fungal Pathogen Fusarium graminearum Using Oxford Nanopore Technology.

Fusarium graminearum is a plant pathogen of global importance which causes not only significant yield loss but also crop spoilage due to mycotoxins that render grain unsafe for human or livestock consumption. Although the full genome of several F. graminearum isolates from different parts of the world have been sequenced, there are no similar studies of isolates originating from China. The current study sought to address this by sequencing the F. graminearum isolate FG-12, which was isolated from the roots of maize seedlings exhibiting typical symptoms of blight growing in the Gansu province, China, using Oxford Nanopore Technology (ONT). The FG-12 isolate was found to have a 35.9 Mb genome comprised of five scaffolds corresponding to the four chromosomes and mitochondrial DNA of the F. graminearum type strain, PH-1. The genome was found to contain an approximately 2.23% repetitive sequence and encode 12,470 predicted genes. Additional bioinformatic analysis identified 437 genes that were predicted to be secreted effectors, one of which was confirmed to trigger a hypersensitive responses (HR) in the leaves of Nicotiana benthamiana during transient expression experiments utilizing agro-infiltration. The F. graminearum FG-12 genome sequence and annotation data produced in the current study provide an extremely useful resource for both intra- and inter-species comparative analyses as well as for gene functional studies, and could greatly advance our understanding of this important plant pathogen.

Extraction and detection of guanosine 5'-diphosphate-3'-diphosphate in amino acid starvation cells of Clavibacter michiganensis.

Guanosine 5'-diphosphate-3'-diphosphate (ppGpp) is a small molecule nucleotide alarmone that can accumulate under the amino acid starvation state and trigger the stringent response. This study reported the extraction of ppGpp from the Gram-positive bacteria Clavibacter michiganensis through methods using formic acid, lysozyme, or methanol. Following extraction, ppGpp was detected through ultra-high-performance liquid chromatography (UHPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The methanol method showed the highest extraction efficiency for ppGpp among the three methods tested. C. michiganensis cells in exponential growth phase was induced in amino acid starvation by serine hydroxamate (SHX) and used for ppGpp extraction and detection. When using the methanol extraction method, the results showed that ppGpp concentrations in SHX-treated samples were 15.645 nM, 17.656 nM, 20.372 nM, and 19.280 nM at 0 min, 15 min, 30 min and 1 h, respectively, when detected using LC-MS/MS. This is the first report on ppGpp extraction and detection in Clavibacter providing a new idea and approach for nucleotide detection and extraction in bacteria.

Role of Penicillin-Binding Proteins in the Viability, Morphology, Stress Tolerance, and Pathogenicity of Clavibacter michiganensis.

Previous research has shown that penicillin-binding proteins (PBPs), enzymes involved in peptidoglycan (PG) assembly, could play an important role during the induction of the viable but nonculturable (VBNC) state, which allows non-spore-forming bacteria to survive adverse environmental conditions. The current study found that Clavibacter michiganensis has seven PBPs. Mutant analysis indicated that deletion of either of the class B PBPs was lethal and that the class A PBPs had an important role in PG synthesis, with the ΔpbpC mutant having an altered cellular morphology that resulted in longer cells that were swollen at one end and had thinner cell walls. The ΔpbpC mutant was also found to produce mucoid colonies in solid culture and a lower final cell titer in liquid medium, as well as having high sensitivity to osmotic stress and lysozyme treatment and surprisingly high pathogenicity. The double mutant, ΔdacB/ΔpbpE, also had a slightly altered phenotype, resulting in longer cells. Further analysis revealed that both mutants had high sensitivity to copper, which resulted in quicker induction into the VBNC state. However, only the ΔpbpC mutant had significantly reduced survivorship in the VBNC state. The study also confirmed that the VBNC state significantly improved the survivorship of wild-type C. michiganensis cells in response to environmental stresses and systemically demonstrated the protective role of the VBNC state in C. michiganensis, which is an important finding regarding its epidemiology and has serious implications for disease management.

Corrigendum: Characterization of cmcp Gene as a Pathogenicity Factor of Ceratocystis manginecans.

[This corrects the article DOI: 10.3389/fmicb.2020.01824.].

Characterization of cmcp Gene as a Pathogenicity Factor of Ceratocystis manginecans.

Ceratocystis manginecans causes mango wilt with significant economic losses. In the infection court, cerato-platanin (CP) family proteins (CPPs) are believed to involve in pathogenesis but has not been determined in C. manginecans. To confirm this function, a CP protein (CmCP) of C. manginecans was characterized in this study. A protoplast of C. manginecans was prepared by treating its mycelia with driselase and lysing enzymes. The cmcp gene was edited using CRISPR/Cas-U6-1 expression vectors in 60% PEG and 50 µg/mL hygromycin B in the medium, resulting in mutants with cmcp deletion (Δcmcp). A complemented mutant (Δcmcp-C) was obtained by transforming cmcp to Δcmcp. Both Δcmcp and Δcmcp-C were characterized by comparing them with a wild-type strain on morphology, mycelial growth, conidial production and pathogenicity. Additionally, cmcp was transformed and expressed in Pichia pastoris, and the derived recombinant protein CmCP caused a severe necrosis on Nicotiana tabacum leaves. CmCP-treated plant leaves showed symptoms of hypersensitive response including electrolyte leakage, reactive oxygen species generation and overexpression of defense-related genes PR-1, PAD3, ERF1, HSR203J, and HIN1. All those results suggested that cmcp gene was required for the growth development of C. manginecans and functioned as a major pathogenicity factor in mango infection.

Allyl Isothiocyanate (AITC) Triggered Toxicity and FsYvc1 (a STRPC Family Member) Responded Sense in Fusarium solani.

Allyl isothiocyanate (AITC) is a natural product used as a food additive. Due to its strong volatility and broad biological activity, AITC is considered as a bio-fumigant to control soil-borne fungal diseases in agriculture, creating an urgent need for evaluation of the antifungal activity of AITC. Here we study the effect of AITC on Fusarium solani growth and explore the molecular mechanisms. The results indicated that AITC causes rapid inhibition of F. solani after 5 min, hyphal deformity, and electrolyte leakage. A yeast-like vacuolar transient receptor potential channel regulator (FsYvc1, a STRPC family member) was identified in F. solani that seems to play a role in this fungi AITC sensitivity. Genetic evidence suggests the gene FsYvc1 is involved in F. solani growth, development, and pathogenicity. Loss of FsYvc1 resulted in hypersensitivity of F. solani to AITC and induced reactive oxygen species (ROS) accumulation ∼ 1.3 to 1.45- folds that of the wild type (WT), and no difference responses to CaCl2, NaCl, KCl, SDS, and Congo red when compared with WT. In addition, ΔFsYvc1-17 showed significantly reduced (∼ 1-fold) glutathione-S-transferase (GST) expression compared with the WT without AITC induction. Upon exposure to 4.8 µg/mL AITC for 3 h, the relative expression levels were ∼ 12-30 fold higher in both the WT and ΔFsYvc1-17. Nevertheless, no difference in GST expression level was observed between the WT and ΔFsYvc1-17. The current study provides novel insights into the toxicity mechanisms of AITC. Considering our results that show the key role of FsYvc1, we propose that it could act as a new molecular target for future fungicide development.

CsIVP functions in vasculature development and downy mildew resistance in cucumber.

Domesticated crops with high yield and quality are frequently susceptible to pathogen attack, whereas enhancement of disease resistance generally compromises crop yield. The underlying mechanisms of how plant development and disease resistance are coordinately programed remain elusive. Here, we showed that the basic Helix-Loop-Helix (bHLH) transcription factor Cucumis sativus Irregular Vasculature Patterning (CsIVP) was highly expressed in cucumber vascular tissues. Knockdown of CsIVP caused severe vasculature disorganization and abnormal organ morphogenesis. CsIVP directly binds to vascular-related regulators YABBY5 (CsYAB5), BREVIPEDICELLUS (CsBP), and AUXIN/INDOLEACETIC ACIDS4 (CsAUX4) and promotes their expression. Knockdown of CsYAB5 resulted in similar phenotypes as CsIVP-RNA interference (RNAi) plants, including disturbed vascular configuration and abnormal organ morphology. Meanwhile, CsIVP-RNAi plants were more resistant to downy mildew and accumulated more salicylic acid (SA). CsIVP physically interacts with NIM1-INTERACTING1 (CsNIMIN1), a negative regulator in the SA signaling pathway. Thus, CsIVP is a novel vasculature regulator functioning in CsYAB5-mediated organ morphogenesis and SA-mediated downy mildew resistance in cucumber.