Verification of the Interaction Target Protein of the Effector ApCE22 of Arthrinium phaeospermum in Bambusa pervariabilis × Dendrocalamopsis grandis.

The study of interaction proteins of the pathogen A. phaeospermum effector protein is an important means to analyze the disease-resistance mechanism of Bambusa pervariabilis × Dendrocalamopsis grandis shoot blight. To obtain the proteins interacting with the effector ApCE22 of A. phaeospermum, 27 proteins interacting with the effector ApCE22 were initially identified via a yeast two-hybrid assay, of which four interaction proteins were obtained after one-to-one validation. The B2 protein and the chaperone protein DnaJ chloroplast protein were then verified to interact with the ApCE22 effector protein by bimolecular fluorescence complementation and GST pull-down methods. Advanced structure prediction showed that the B2 protein contained the DCD functional domain related to plant development and cell death, and the DnaJ protein contained the DnaJ domain related to stress resistance. The results showed that both the B2 protein and DnaJ protein in B. pervariabilis × D. grandis were the target interaction proteins of the ApCE22 effector of A. phaeospermum and related to the stress resistance of the host B. pervariabilis × D. grandis. The successful identification of the pathogen effector interaction target protein in B. pervariabilis × D. grandis plays an important role in the mechanism of pathogen-host interaction, thus providing a theoretical basis for the control of B. pervariabilis × D. grandis shoot blight.

Identification of the interacting proteins of Bambusa pervariabilis × Dendrocalamopsis grandis in response to the transcription factor ApCtf1ß in Arthrinium phaeospermum.

Arthrinium phaeospermum is the main pathogen that causes Bambusa pervariabilis × Dendrocalamopsis grandis blight. It secretes the cutinase transcription factor ApCtf1ß, which has been shown to play an important role in B. pervariabilis × D. grandis virulence. However, knowledge about the interaction target genes of ApCtf1ß in B. pervariabilis × D. grandis remains limited. A cDNA library for the yeast two-hybrid system was constructed from B. pervariabilis × D. grandis shoots after 168 h treatment with A. phaeospermum. The library was identified as 1.20 × 107 cfu, with an average insert >1,000 bp in size and a 100% positive rate, providing a database for the subsequent molecular study of the interaction between A. phaeospermum and B. pervariabilis × D. grandis. The yeast two-hybrid (Y2H), bimolecular fluorescence complementation (BiFC), and glutathione-S-transferase (GST) pull-down assays were used to screen for and identify two ApCtf1ß interacting target proteins, BDUbc and BDSKL1, providing a reliable theoretical basis to study the molecular mechanism underlying B. pervariabilis × D. grandis resistance in response to A. phaeospermum, which would, in turn, establish a platform to develop new strategies for the sustainable and effective control of the blight diseases of forest trees.

Functional Identification of Arthrinium phaeospermum Effectors Related to Bambusa pervariabilis × Dendrocalamopsis grandis Shoot Blight.

The shoot blight of Bambusa pervariabilis × Dendrocalamopsis grandis caused by Arthrinium phaeospermum made bamboo die in a large area, resulting in serious ecological and economic losses. Dual RNA-seq was used to sequence and analyze the transcriptome data of A. phaeospermum and B. pervariabilis × D. grandis in the four periods after the pathogen infected the host and to screen the candidate effectors of the pathogen related to the infection. After the identification of the effectors by the tobacco transient expression system, the functions of these effectors were verified by gene knockout. Fifty-three differentially expressed candidate effectors were obtained by differential gene expression analysis and effector prediction. Among them, the effectors ApCE12 and ApCE22 can cause programmed cell death in tobacco. The disease index of B. pervariabilis × D. grandis inoculated with mutant ΔApCE12 and mutant ΔApCE22 strains were 52.5% and 47.5%, respectively, which was significantly lower than that of the wild-type strains (80%), the ApCE12 complementary strain (77.5%), and the ApCE22 complementary strain (75%). The tolerance of the mutant ΔApCE12 and mutant ΔApCE22 strains to H2O2 and NaCl stress was significantly lower than that of the wild-type strain and the ApCE12 complementary and ApCE22 complementary strains, but there was no difference in their tolerance to Congo red. Therefore, this study shows that the effectors ApCE12 and ApCE22 play an important role in A. phaeospermum virulence and response to H2O2 and NaCl stress.

Comparative Transcriptomics and Gene Knockout Reveal Virulence Factors of Arthrinium phaeospermum in Bambusa pervariabilis × Dendrocalamopsis grandis.

Arthrinium phaeospermum can cause branch wilting of Bambusa pervariabilis × Dendrocalamopsis grandis, causing great economic losses and ecological damage. A. phaeospermum was sequenced in sterile deionized water (CK), rice tissue (T1) and B. pervariabilis × D. grandis (T2) fluid by RNA-Seq, and the function of Ctf1ß 1 and Ctf1ß 2 was verified by gene knockout. There were 424, 471 and 396 differentially expressed genes between the T2 and CK, T2 and T1, and CK and T1 groups, respectively. Thirty DEGs had verified the change in expression by fluorescent quantitative PCR. Twenty-nine DEGs were the same as the expression level in RNA-Seq. In addition, ΔApCtf1ß 1 and ΔApCtf1ß 2 showed weaker virulence by gene knockout, and the complementary strains Ctf1ß 1 and Ctf1ß 2 showed the same virulence as the wild-type strains. Relative growth inhibition of ΔApCtf1ß 1 and ΔApCtf1ß was significantly decreased by 21.4% and 19.2%, respectively, by adding H2O2 compared to the estimates from the wild-type strain and decreased by 25% and 19.4%, respectively, by adding Congo red. The disease index of B. pervariabilis × D. grandis infected by two mutants was significantly lower than that of wild type. This suggested that Ctf1ß genes are required for the stress response and virulence of A. phaeospermum.

Differential transcriptome analysis and identification of genes related to resistance to blight in three varieties of Bambusa pervariabilis × Dendrocalamopsis grandis.

BACKGROUND: Bambusa pervariabilis × Dendrocalamopsis grandis is a fast-growing bamboo that is widely introduced in southern China and has great economic and ecological benefits. In recent years, a blight of B. pervariabilis × D. grandis caused by Arthrinium phaeospermum has led to much branch damage and even death of entire bamboo forests. METHODS: To screen for resistance genes in B. pervariabilis × D. grandis, transcriptome sequencing technology was used to compare the gene expression profiles of different varieties of B. pervariabilis × D. grandis with variable resistance and the same varieties under different treatments. The Clusters of Orthologous Groups of Proteins (COG) database; the Gene Ontology (GO) database; and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database were used to annotate and analyse the differentially expressed genes. RESULTS: A total of 26,157 and 11,648 differentially expressed genes were obtained in the different varieties after inoculation with A. phaeospermum and the same varieties after inoculation A. phaeospermum or sterile water, respectively. There were 23 co-upregulated DGEs and 143 co-downregulated DEGs in #3 and #8, #6 and #8, #6 and #3. There were 50 co-upregulated DGEs and 24 co-downregulated DEGs in the same varieties after inoculation A. phaeospermum or sterile water. The results showed that many genes involved in cell wall composition synthesis, redox reactions and signal transduction were significantly different after pathogen infection. Twenty-one candidate genes for blight resistance, such as pme53, cad5, pod, gdsl-ll and Myb4l, were found. The qRT-PCR results were consistent with the sequencing results, verifying their authenticity. These results provide a foundation for the further exploration of resistance genes and their functions.

First Report of Arthrinium kogelbergense Causing Blight Disease of Bambusa intermedia in Sichuan Province, China.

Bambusa intermedia Hsueh et Yi, a species in the Gramineae family, is mainly distributed in southern China and is commonly found in the Sichuan, Yunnan, and Guangdong Provinces and in the Guangxi Autonomous Region. It is economically significant as a building material, a food source, and for applications in various other raw products. In July 2017, a blight disease was found on B. intermedia stems, affecting approximately 40% of 9600 plants in the Changning and Jiangan counties of Sichuan Province, China. In the early stages of the disease, leaves at the top of the plants withered and yellowed, eventually falling off. Subsequently, the stems and upper branches discolored and were dead to varying degrees. To isolate the causal fungus, 100 samples from B. intermedia culms and branches were collected from symptomatic plants in Changning and Jiangan counties. Small sections (4 to 5 mm2) were surface-sterilized for 30 s in 3% sodium hypochlorite and 60 s in 75% ethanol. The samples were then rinsed three times in sterile water, placed onto potato dextrose agar (PDA) amended with streptomycin sulfate (50 µg/ml, Solarbio, Beijing), and incubated in a dark incubator at 25 ± 1°C for 6 days. A total of 120 isolates were obtained, of which 89 isolates had similar colony morphology and conidial measurements. Colony characteristics of the fungus on the PDA surface were dirty white, reverse dirty white to pale luteous, colonies flat, floccose, with moderate aerial mycelium. Hyphae were smooth, hyaline, branched, and septate. Conidiophores were erect, septate, pale brown, smooth, and reduced to conidiogenous cells. Conidiogenous cells were pale brown, smooth, and ampulliform. Conidia were 5.69-8.03 × 4.32-7.30 µm (30 conidia per isolate, Leica Application Suite X 3.4.1.17822) single celled, brown to black, smooth, and globose to ellipsoid shape, mostly globose in surface view and lenticular in side view with an equatorial ring. Based on morphological characteristics, it was identified as an Arthrinium species (Crous and Groenwald 2013). PCRs were performed with primers ITS4/ITS5 for the ITS region (White et al. 1990), primers T1/Bt2b for the ß-tubulin gene (TUB) (O'Donnell and Cigelnik 1997, Glass and Donaldson 1995) , primers 5.8sR/LR5 (Vilgalys and Hester 1990) and LR0R/TW13 (Hamayun et al. 2009) for 28S large-subunit (LSU) rDNA. Newly generated representative sequences were deposited in GenBank, ITS sequence (GenBank No. MT415395), TUB sequence (MT415365), and LSU sequence (MT415396, MT415398). Comparison to ex-type isolate sequences showed strong homology with Arthrinium kogelbergense Crous (CBS 113333, Crous and Groenwald 2013). ITS: 99.65% identity to Accession NR_120272; TUB: 99.75% identity to Accession KF144984; LSU: 99.60% and 100.00% identity to Accession NG_042779. To confirm its pathogenicity and to fulfill Koch's postulates, 20 one-year-old B. intermedia plants were wounded with a sterile needle at a depth of 1 mm in the stems and twigs, and were inoculated with a 5-mm-diameter disk of PDA that was colonized by the isolates (2 plants per isolate in each inoculation experiment, 10 repetitions) (Li et al. 2016). Ten control plants were treated similarly except that they were mock inoculated with PDA plugs without the fungus. All plants were kept at 25-28°C and covered with plastic bags to maintain high relative humidity (90-95%) on a 12-h light/dark incubation. Thirty days later, the inoculated plants showed the same symptoms observed originally, and the controls remained healthy. The same fungus was reisolated from the infected stems and twigs and showed similar morphological characteristics and molecular traits. To our knowledge, this is the first report of A. kogelbergense as a causal agent of blight disease on B. intermedia in Sichuan Province, China.

Whole genome sequence of Diaporthe capsici, a new pathogen of walnut blight.

Many fungi in the Diaporthe genus across the world are pathogenic. Diaporthe capsici. is a pathogenic fungus that can infect peppers and walnuts, causing their death. The aim of this study was to develop a genomic resource to provide substantial data and a theoretical basis for research on molecular pathogenesis, transcriptome, proteome, and metabonome of D. capsici. The whole genome of D. capsici was sequenced using the PacBio RSII sequencing platform, and functional annotation was performed using different public databases. The genome was found to be 57.56 Mb in size, with an N50 contig size of 5,171,887 bp, and encodes 14,425 putative genes. This is the first genome-scale assembly and annotation for D. capsici, which is the eighth species in Diaporthe to be sequenced.

Whole-genome sequence of Arthrinium phaeospermum, a globally distributed pathogenic fungus.

Arthrinium phaeospermum (Corda) M.B. Ellis is a globally distributed pathogenic fungus with a wide host range; its hosts include not only plants, but also humans and animals. This study aimed to develop genomic resources for A. phaeospermum to provide solid data and a theoretical basis for further studies of its pathogenesis, transcriptomics, proteomics, metabolomics and RNA genomics. The genome was obtained from the mycelia of the strain AP-Z13 using a combination of analyses with the high-throughput Illumina HiSeq 4000 system and PacBio RSII LongRead sequencing platform. Functional annotation was performed by BLASTing protein sequences against those in different publicly available databases to obtain their corresponding annotations. The genome is 48.45 Mb in size, with an N90 scaffold size of 1,931,147 bp, and encodes 19,836 putative predicted genes. This is the first report of the genome-scale assembly and annotation for A. phaeospermum, the first species in the genus Arthrinium to be subjected to whole genome sequencing.

Comparative transcriptome analysis of Bambusa pervariabilis × Dendrocalamopsis grandis against Arthrinium phaeospermum under protein AP-toxin induction.

Bambusapervariabilis × Dendrocalamopsisgrandis, a fast-growing and easily propagated bamboo species, has been extensively planted in the southern China, resulting in huge ecological benefits. In recent years, it was found that the pathogenic fungus Arthrinium phaeospermum caused the death of a large amount of bamboo. In this study, the transcriptome of B. pervariabilis × D. grandis, induced by inactivated protein AP-toxin from A. phaeospermum was sequenced and analyzed, to reveal the resistance mechanism induced by biotic agents of B. pervariabilis × D. grandis against A. phaeospermum at the gene level. Transcriptome sequencing was performed by Illumina HiSeq 2000 in order to analyze the differentially expressed genes (DEGs) of B. pervariabilis × D. grandis in response to different treatment conditions. In total, 201,875,606 clean reads were obtained, and the percentage of Q30 bases in each sample was more than 94.21%. There were 6398 DEGs in the D-J group (inoculation with a pathogenic spore suspension after three days of AP-toxin induction) compared to the S-J group (inoculation with a pathogenic spore suspension after inoculation of sterile water for three days) with 3297 up-regulated and 3101 down-regulated genes. For the D-S group (inoculation with sterile water after inoculation of AP-toxin for three days), there were 2032 DEGs in comparison to the S-S group (inoculation with sterile water only), with 1035 up-regulated genes and 997 down-regulated genes. These identified genes were mainly involved in lignin and phytoprotein synthesis, tetrapyrrole synthesis, redox reactions, photosynthesis, and other processes. The fluorescence quantitative results showed that 22 pairs of primer amplification products were up-regulated and 7 were down-regulated. The rate of similarity between these results and the sequencing results of the transcription group was 100%, which confirmed the authenticity of the transcriptome sequencing results. Redox proteins, phenylalanine ammonia lyase, and S-adenosine-L-methionine synthetase, among others, were highly expressed; these results may indicate the level of disease resistance of the bamboo. These results provide a foundation for the further exploration of resistance genes and their functions.

Differential Proteome Analysis of Hybrid Bamboo (Bambusa pervariabilis × Dendrocalamopsis grandis) Under Fungal Stress (Arthrinium phaeospermum).

In this study, TMT (tandem mass tag)-labeled quantitative protein technology combined with LC-MS/MS (liquid chromatography-mass spectrometry/mass spectrometry) was used to isolate and identify the proteins of the hybrid bamboo (Bambusa pervariabilis × Dendrocalamopsis grandis) and the bamboo inoculated with the pathogenic fungi Arthrinium phaeospermum. A total of 3320 unique peptide fragments were identified after inoculation with either A. phaeospermum or sterile water, and 1791 proteins were quantified. A total of 102 differentially expressed proteins were obtained, of which 66 differential proteins were upregulated and 36 downregulated in the treatment group. Annotation and enrichment analysis of these peptides and proteins using the GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) databases with bioinformatics software showed that the differentially expressed protein functional annotation items were mainly concentrated on biological processes and cell components. The LC-PRM/MS (liquid chromatography-parallel reaction monitoring/mass spectrometry) quantitative analysis technique was used to quantitatively analyze 11 differential candidate proteins obtained by TMT combined with LC-MS/MS. The up-down trend of 10 differential proteins in the PRM results was consistent with that of the TMT quantitative analysis. The coincidence rate of the two results was 91%, which confirmed the reliability of the proteomic results. Therefore, the differentially expressed proteins and signaling pathways discovered here may be the further concern for the bamboo-pathogen interaction studies.

Differential Proteomics Based on TMT and PRM Reveal the Resistance Response of Bambusa pervariabilis × Dendrocalamopisis grandis Induced by AP-Toxin.

Bambusa pervariabilis McClure × Dendrocalamopsis grandis (Q.H.Dai & X.l.Tao ex Keng f.) Ohrnb. blight is a widespread and dangerous forest fungus disease, and has been listed as a supplementary object of forest phytosanitary measures. In order to study the control of B. pervariabilis × D. grandis blight, this experiment was carried out. In this work, a toxin purified from the pathogen Arthrinium phaeospermum (Corda) Elli, which causes blight in B. pervariabilis × D. grandis, with homologous heterogeneity, was used as an inducer to increase resistance to B. pervariabilis × D. grandis. A functional analysis of the differentially expressed proteins after induction using a tandem mass tag labeling technique was combined with mass spectrometry and liquid chromatography mass spectrometry in order to effectively screen for the proteins related to the resistance of B. pervariabilis × D. grandis to blight. After peptide labeling, a total of 3320 unique peptides and 1791 quantitative proteins were obtained by liquid chromatography mass spectrometry analysis. Annotation and enrichment analysis of these peptides and proteins using the Gene ontology and Kyoto Encyclopedia of Genes and Genomes databases with bioinformatics software show that the differentially expressed protein functional annotation items are mainly concentrated on biological processes and cell components. Several pathways that are prominent in the Kyoto Encyclopedia of Genes and Genomes annotation and enrichment include metabolic pathways, the citrate cycle, and phenylpropanoid biosynthesis. In the Protein-protein interaction networks four differentially expressed proteins-sucrose synthase, adenosine triphosphate-citrate synthase beta chain protein 1, peroxidase, and phenylalanine ammonia-lyase significantly interact with multiple proteins and significantly enrich metabolic pathways. To verify the results of tandem mass tag, the candidate proteins were further verified by parallel reaction monitoring, and the results were consistent with the tandem mass tag data analysis results. It is confirmed that the data obtained by tandem mass tag technology are reliable. Therefore, the differentially expressed proteins and signaling pathways discovered here is the primary concern for subsequent disease resistance studies.

Metabolomics responses of Bambusa pervariabilis × Dendrocalamopsis grandis varieties to Biotic (pathogenic fungus) stress.

Bambusa pervariabilis × Dendrocalamopsis grandis blight, caused by Arthrinium phaeospermum, is one of the most common and serious diseases in bamboo and occurs in the newly born twigs. Bamboo has suffered large dead areas, including more than 3000 hm2, which greatly threatens the process of returning farmlands to forests and the construction of ecological barriers. To identify differential metabolites and metabolic pathways associated with B. pervariabilis × D. grandis to A. phaeospermum, ultra-performance liquid chromatography (UPLC) and quadrupole-time of flight (Q-TOF) Mass Spectrometry (MS) combined with a data-dependent acquisition method was used to analyse the entire sample spectrum. In total, 13223 positive ion peaks and 10616 negative ion peaks were extracted. OPLS-DA and several other analyses were performed using the original data. The OPLS-DA models showed good quality and had strong predictive power, indicating clear trends in the analyses of the treatment and control groups. Clustering and KEGG pathway analyses were used to screen the differential metabolites in the treatment and control groups from the three B. pervariabilis × D. grandis varieties and reflected their metabolic responses induced by A. phaeospermum infection. The results showed that the three B. pervariabilis × D. grandis varieties mode showed significant changes in the following six resistance-related metabolites after A. phaeospermum invasion in positive and negative ion modes: proline, glutamine, dictamnine, apigenin 7-O-neohesperidoside, glutamate, and cis-Aconitate. The following four main metabolic pathways are involved: Arginine and proline metabolism, Glyoxylate and dicarboxylate metabolism, Biosynthesis of alkaloids derived from shikimate pathway, and Flavone and flavonol biosynthesis. This study lays a foundation for the later detection of differential metabolites and metabolic pathways for targeting, and provides a theoretical basis for disease-resistant breeding and the control of B. pervariabilis × D. grandis blight.

Screening of Endophytic Antagonistic Bacterium from Phellodendron amurense and Their Biocontrol Effects against Canker Rot.

Thirty-four strains of bacteria were isolated from Phellodendron amurense. Using Nectria haematococca as an indicator strain, the best strain, B18, was obtained by the growth rate method. The morphological, physiological and biochemical characteristics of strain B18 and its 16S DNA gene sequence were identified, and the biocontrol effect of strain B18 was assessed in pot and field tests, as well as in a field-control test. Drilling methods were used to determine the antibacterial activity of metabolites from strain B18 and their effects on the growth of pathogen mycelia and spores. The best bacteriostatic rate was 85.4%. B18 can hydrolyse starch and oxidize glucose but does not produce gas; a positive result was obtained in a gelatine liquefaction test. According to 16S DNA gene sequencing, strain B18 is Bacillus methylotrophicus (GenBank accession number: MG457759). The results of pot and field-control trials showed 98% disease control when inoculating 108 cfu/ml of the strain. The disease control effect of the B18 culture liquid (concentrations of 108, 2 × 106, 106, 5 × 105 and 2.5 × 105 cfu/ml) in the field-control test was higher than 80%, and the cure rate of the original delivery solution was 96%. Therefore, in the practical forestry production, a 2.5 × 105 cfu/ml culture liquidshould be applied in advance to achieve good control effects.

Acetylbritannilactone suppresses growth via upregulation of krüppel-like transcription factor 4 expression in HT-29 colorectal cancer cells.

Acetylbritannilactone (ABL) is a new active compound isolated from Inula Britannica L, a traditional Chinese medicinal herb. It has been reported that ABL can inhibit the proliferation of vascular smooth muscle cells (VSMCs) and neointima formation after balloon injury in rats. ABL also shows chemopreventive properties by inducing cell apoptosis in breast and ovarian cancers, but the antitumor activity and the molecular targets of ABL in colon cancer cells have not been determined. In this study, we showed that ABL inhibits the growth in dose- and time-dependent manners by inducing cell cycle arrest in G0/G1 phase of HT-29 human colon cancer cells. This suppression was accompanied by a strong decrease of cyclin E and CDK4 protein levels, and an increase in p21 protein expression in HT-29 cells. We also show that ABL-induced growth inhibition is associated with the upregulation of KLF4 expression. The overexpression of KLF4 by infection with pAd-KLF4 resulted in growth inhibition, with decrease in the protein levels of cyclin E and CDK4, and increase in the expression of p21, similarly to the effects of ABL. Conversely, knockdown of KLF4 using a specific siRNA impaired the ABL-induced growth inhibition in HT-29 cells. These results suggest that KLF4 as an important cellular target of ABL mediates the growth inhibition of HT-29 cells induced by ABL via upregulation of p21 expression.

miR-146a and Krüppel-like factor 4 form a feedback loop to participate in vascular smooth muscle cell proliferation.

MicroRNAs are phenotypic regulators of vascular smooth muscle cells (VSMCs). In this paper, we demonstrate that miR-146a targets the Krüppel-like factor 4 (KLF4) 3'-untranslated region and has an important role in promoting VSMC proliferation in vitro and vascular neointimal hyperplasia in vivo. Silencing of miR-146a in VSMCs increases KLF4 expression, whereas overexpression of miR-146a decreases KLF4 levels. Furthermore, we demonstrate that KLF4 competes with Krüppel-like factor 5 (KLF5) to bind to and regulate the miR-146a promoter, and that KLF4 and KLF5 exert opposing effects on the miR-146a promoter. Overexpression of KLF4 in VSMCs decreases miR-146a transcription levels. By using both gain-of-function and loss-of-function approaches, we found that miR-146a promotes VSMC proliferation in vitro. Transfection of antisense miR-146a oligonucleotide into balloon-injured rat carotid arteries markedly decreased neointimal hyperplasia. These findings suggest that miR-146a and KLF4 form a feedback loop to regulate each other's expression and VSMC proliferation.