Fusarium sacchari is one of the major pathogenic fungus that cause sugarcane pokkah boeng. In order to explore the function of metalloproteinase effector proteins in F. sacchari, we used the genomic data of F. sacchari to predict the secretory metalloproteinase proteins, and successfully amplified a zinc-type metalloproteinase effector protein gene Fs03538. The results showed that Fs03538 contained a typical ZnMc super family domain, and the 1-18 amino acid sequences at the N-terminal of the protein contain specific signal sequences. Subcellular localization showed that Fs03538 could be localized in the nucleus of Nicotiana benthamiana; qRT-PCR analysis showed that the expression level of Fs03538 was induced and reached the highest peak at 12 h post F. sacchari infection. Agrobacterium tumefaciens mediated transient expression system confirmed that Fs03538 could inhibit the necrosis of tobacco cells induced by mouse Bcl-2-associated X protein (BAX). As compared with the wild type F. sacchari strain CNO-1, the Fs03538 knock-out mutant showed no significant difference on mycelial growth and conidia production, but the pathogenicity on sugarcane was significantly decreased. Taken together, the results of the study suggest that Fs03538 is an important virulence factor of F. sacchari, which highly expressed at the time of infection and could inhibit the host immune responses by entering the host cell nucleus.

Fusarium solani, known for its extensive host range, is the causal agent of the destructive root rot disease in agriculture production. Secreted proteins play important roles in the infection of host plants by phytopathogenic fungi. To identify the secreted proteins and effectors in F. solani, we performed an in-depth analysis of the F. solani genome in this study. Among the total 17654 genomic proteins, 1032 proteins were predicted to be the candidate secreted proteins by using SignalP, TMHMM, WoLF PSORT and PredGPI softwares, accounting for 5.85% of the total proteins in F. solani. Among them, 258 proteins were predicated to be carbohydrate-active enzymes (CAZymes) by using the dbCAN3 software, with the glycoside hydrolase family being the most abundant. Furthermore, 185 secreted proteins were predicated to be candidate effectors, with 183 sequences being annotated in the PHI database. By employing a virus-based transient expression system, we investigated the effect of the 5 candidate effectors annotated for increased virulence on BAX-triggered programmed cell death, and the result showed that the two effectors (XP_046140852.1 and XP_046131041.1) could suppress BAX-triggered programmed cell death in N. benthamiana. These findings provide not only an important reference for further analysis of the pathogenic molecular mechanism of F. solani but also a theoretical basis for understanding the interactions between F. solani and host plants.

Rice is an important grain crop in the world. Although some genes that confer resistance to rice blast and bacterial blight, two important diseases threatening rice production, have been identified in rice plants, the corresponding resistance gene resources remain scarce. In this study, we found that the transcription factor OsEIL4 is involved in regulating rice resistance to these two rice diseases. Quantitative real-time PCR (qPCR) assays showed that the expression of OsEIL4 was markedly induced upon Magnaporthe oryzae (M. oryzae) or Xanthomonas oryzae pv. oryzae (Xoo) infection. Moreover, compared with wild-type rice plants, Oseil4 (CRISPR/Cas9-based OsEIL4 knockout) and OsEIL4-RNAi rice lines were more susceptible, while OsEIL4-OX (overexpression) plants were more resistant to M. oryzae and Xoo. Further qPCR analysis of the transcript levels of the marker genes of ethylene pathway and defense-related genes OsPR1a and OsPR5 exhibited that they were downregulated in Oseil4 lines but upregulated in OsEIL4-OX lines, suggesting that OsEIL4, which functions as a positive regulator in ethylene pathway, mediates rice resistance by modulating PR genes expression. Subcellular localization and yeast-one-hybrid assay results confirmed that OsEIL4 has transcriptional activity, indicating that it may regulate rice disease resistance by exercising transcriptional regulatory function. This study explores a gene resource with broad-spectrum resistance, providing a new possibility for molecular breeding of rice disease resistance.

In order to clarify the protein-protein interaction of Botrytis cinerea and further explore the molecular mechanism of B. cinerea pathogenicity, a protein-protein interaction network (PPI) map of B. cinerea containing 2 296 proteins and 9 376 pairs of interactions was constructed by homology mapping (Interolog) method. The domain-domain interactions (Domain-domain interactions) method was used to further screen and optimize the protein-protein interaction network map of B. cinerea. A high-confidence protein-protein interaction network containing 1 233 proteins and 2 585 pairs of interactions was constructed. The network diagram was divided into 27 functional modules by MCODE algorithm, and the interaction protein subnetwork of known pathogenic proteins GB1, RAS2, BMP1, and BMP3 were analyzed. The molecular mechanism of its pathogenicity was predicted. The interaction between BofuT4_P103090 and BofuT4_P056160 and BofuT4_P007800 proteins in the protein interaction network was verified by yeast two-hybrid technique. The interaction between BofuT4_P103090 and BofuT4_P056160 and BofuT4_P007800 proteins was determined, and the reliability of the protein-protein interaction network constructed in this study was verified. The results of this study laid a foundation for elucidating the molecular mechanism of B. cinerea pathogenesis, and provided a reference for the study of protein-protein interaction networks and pathogenesis of other species.

Pythium myriotylum is a soil-borne oomycete that can infect a wide range of economically important crops such as soybeans, tomatoes and wheat. It causes root rot and stem base rot resulting in serious harm. In this study, we identified and characterized a carbonic anhydrase family protein-PmCA1 in P. myriotylum. We found that PmCA1 was upregulated during plant infection. In addition, PmCA1 inhibited flg22 induced reactive oxygen species burst and defense related gene expression, promoting the infection of Phytophthora capsici. These results indicate that PmCA1 may be a virulence factor of P. myriotylum, promoting pathogen colonization by inhibiting plant PTI immune response. Taken together, our results provide a basis for the functional study of the secreted proteins by P. myriotylum, as well as theoretical support for exploring the interaction between P. myriotylum and host plants.

Potato Early Blight (PEB) is an important disease of the foliage of potatoes during the growing season and is widespread in all major potato producing areas worldwide. At present, there are no specific agents and potato varieties resistant to the disease completely. In this study, we identified the main cultivar ‘Cooperation-88’ (C88) in Yunnan Province was resistant to the potato early blight caused by Alternaria solani inoculated on leaves in comparison with the susceptible variety ‘Désirée’ by AUDPC. High-throughput RNA-seq in ‘Cooperation-88’ after infection at the early (A. so_e, 0-72 h), middle (A. so_m, 73-120 h) and late (A. so_l, >120 h) stages was performed on Illumina HiSeq PE150 platform. Transcriptome analysis revealed a total of 13 083 genes expressed differentially at A. so_e, of which 7 438 were up-regulated and 5 645 were down-regulated. At A. so_m, a total of 12 121 genes was differentially expressed, of which 3 299 were up-regulated and 8 822 were down-regulated. At A. so_l, a total of 10 530 genes was differentially expressed, of which 1 686 were up-regulated and 8 844 were down-regulated. A set of 2 720 identical genes was found in all the three periods, while 4 997 genes specific to A. so_e, 3 975 genes specific to A. so_m and 3 230 genes specific to A. so_l. Based on gene ontology (GO) term and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and qRT-PCR validation along with the results of electron microscopic observations, it was speculated that the increased synthesis of pectin lyase and cellulose synthetase was involved in cell wall remodeling of ‘C88’ in the early stage of A. solani infection. The expression levels of glutathione S-transferase and cytochrome P450 were significantly up-regulated in ‘C88’ at mid-infestation, which were involved in the detoxification pathway. Late synthesis of large amounts of antioxidants to stimulate defence mechanisms. In the three periods, high expression levels of transcription factors, such as bHLH, ZIP, MYB, ERF, etc, associated with disease resistance and extensive involvement in the ubiquitination pathway. Overall, our findings could provide a theoretical basis for research on the early blight resistance in potato and accelerate the resistance breeding.

Powdery mildew of rubber tree caused by Oidium heveae is one of the major foliar diseases that affect rubber production, resulting in extensive leaf shedding and significant reduction in latex production. Breeding and planting O. heveae-resistant rubber varieties is the most economic and effective measure for the prevention and control of the disease. Identification of resistant proteins in rubber trees and investigation of the corresponding regulatory networks are therefore of great significance in unraveling the mechanism underlying the resistance response of rubber tree to powdery mildew. In this study, total RNA was extracted from mature rubber leaves (leaves sampled at 48 hpi : leaves sampled at 0 dpi=2:1) inoculated with Oidium heveae HO-1 using the Trizol method. A cDNA library of rubber tree leaves infected with powdery mildew was subsequently constructed using the Gateway technology. The primary library was recombined with pGADT7-DEST and pPR3-N-DEST secondary library vectors through LR recombination, resulting in the construction of hybrid secondary libraries for membrane system and nuclear system yeast, respectively. Both libraries have a capacity of more than 1.0×10^-7 CFU, with a recombination rate of 100%. The length of inserted fragments ranges from 0.1 to 2.0 kb, with an average length of 1.5 kb. Meanwhile, bait vector containing the full-length coding region or the leucine-rich repeat (LRR) domain of the disease-resistant protein HbCNL2 was generated and used as a bait to screen for interacting proteins in the yeast library, and as a result, 7 potentially interacting proteins were obtained. Further yeast two-hybrid point-to-point validation confirmed the interaction between HbKLCR1 or HbGAIP and the LRR domain of HbCNL2. GO (Gene Ontology) annotation analysis demonstrated that HbKLCR1 has protein binding activity and HbGAIP is involved in the gibberellin signaling pathway, suggesting their potential roles in disease resistance responses of rubber trees. These results indicate that the cDNA library constructed in this study has high quality and good integrity, which provides important technical support for the identification of O. heveae-resistant rubber proteins and unraveling the protein interaction networks.

Leucine-rich repeat receptor-like protein kinases (LRR-RLKs), a typical type of receptor-like kinase in plant, play important roles in response to pathogen infection. To clarify basic characteristics of the LRR-RLK family members in foxtail millet and their roles in resistance to infection by Sclerospora graminicola, members of this gene family in foxtail millet were identified, and their evolutionary pattern, sequence characteristics, gene structure, promoter sequence and expression pattern were analyzed by bioinformatics method. Meanwhile, the transcriptome data of resistant and susceptible foxtail millet varieties infected with S. graminicola were obtained at 3 different growth stages, and the co-expression modules of resistance gene and the core genes were identified via the weighted gene co-expression network analysis (WGCNA). The results showed that the LRR-RLK genes were distributed on overall 9 chromosomes of foxtail millet. A phylogenetic analysis was conducted on LRR-RLK genes from foxtail millet and Arabidopsis, and the result indicated that they were mainly divided into 4 categories. Structural analysis displayed that their kinase domains were relatively conservative. The promoter regions of these LRR-RLK genes contained multiple cis-acting elements related to defense and stress responses as well as meristem expression, indicating their involvement in multiple biological processes. A co-expression network of resistance-related genes was developed by using WGCNA. Of 44 gene co-expression modules that were identified, 3 (Turquoise, Blue and Yellow) were specific modules associated with resistance to S. graminicola, from which 12 core genes were identified. Functional annotation showed that these genes were involved in plant disease resistance. Further RT-qPCR analysis of the 6 core genes (Seita.9G413000, Seita.9G296000, Seita.9G557200, Seita.9G493600, Seita.3G241700 and Seita.9G163200) confirmed that they were induced in response to S. graminicola infection, indicating that these core genes may play important roles in resistance to the pathogen infection. The results provide a valuable reference for further revealing the molecular mechanism underlying the resistance of foxtail millet to S. graminicola.

Colletotrichum higginsianum is the major pathogen of anthracnose in cruciferous plants, which can severely damage the production of Chinese flowering cabbage (Brassica parachinensis) in south China. Scytalone dehydratase (Scd) is a key enzyme in the biosynthesis of DHN-melanin that can affect pathogenicity by mediated DHN-melanin biosynthesis in many plant pathogenic fungi. In this study, we identified a conserved scytalone dehydratase ChScd in C. higginsianum. The expression of gene ChSCD was analysed by using RT-qPCR technology, and it was found that the expression level of this gene was significantly up-regulated during the melanization of the hyphae and appressoria of C. higginsianum. Meanwhile, in order to analyse the biological function of the gene ChSCD, the Agrobacterium tumefaciens-mediated transformation technology was used to construct knockout and complementation mutant strains of the gene ChSCD. The results showed that the knockout of gene ChSCD blocked the biosynthesis of DHN-melanin in C. higginsianum, resulting in the loss of the melanization ability in both hyphae and appressoria, and leading to the significantly reduced in tolerance to cell wall interfering substances and oxidative stresses, the appressorium formation rate, turgor pressure, and pathogenicity of the ChscdΔ mutants. In summary, ChScd plays a crucial role in the biosynthesis of DHN melanin in C. higginsianum, which in turn affects the stress resistance, the formation rate of appressorium, turgor pressure, and the pathogenicity of the pathogen.

Most citrus germplasm resources are susceptible to Xanthomonas citri subsp. citri (Xcc), which has seriously hindered the development of the citrus industry in China. In this study, 88 POD genes were identified in the citron genome and divided into 2 groups by phylogenetic tree analysis. Through RNA-seq analysis of the leaves from Citron C-05 and ‘Bingtang’ sweet orange, which are resistant and susceptible to Xcc respectively, 16 POD genes with Xcc-induced expression were obtained. The expression of CmPOD05, which has a high expression level and a large difference in resistant and susceptible germplasm, was further verified by qRT-PCR, and its possible localization to the cytoplasmic membrane was determined by subcellular localization in tobacco. The protein differences of POD05 homologous genes among different citrus germplasm were also analyzed, as well as several disease-resistance-related elements such as defense and stress response regulation through the analysis of cis-acting elements of their promoter sequences. Transient overexpression of CmPOD05 in ‘Bingtang’ sweet orange leaves significantly reduced the growth of Xcc per unit leaf area on the third day after Xcc inoculation compared to the control. Transient overexpression of CmPOD05 in Citron C-05 and tobacco leaves increased the H₂O₂ and superoxide anion content of leaves. The results indicated that CmPOD05 may be involved in the resistance process of Citron C-05 to Xcc.

Meloidogyne enterolobii, which is highly pathogenic to a wide range of host plants and spreads rapidly, can cause devastating damage to many crops. To deeply analyze the pathogenic mechanism of this nematode, here we take T106, a gene specifically induced in tomato roots in response to M. enterolobii infection based on previous transcriptome data, as our target. We silenced T106 in tomato plants via TRV virus-induced gene silencing technology, and then inoculated tomato seedlings with M. enterolobii to observe the difference in nematode and giant cell development in root system between T106-silenced and T106-unsilenced plants. The results showed that the silencing vector we constructed could effectively silence T106 gene in tomato plants, with a silencing efficiency of 85%; compared with T106-unsilenced control plants, there was no significant decrease in the percentage of root knots in T106-silenced plants, but the development of M. enterolobii in root knots was inhibited, and the number of eggs produced by M. enterolobii was reduced by 79.3%; meanwhile, the area occupied by giant cells was also decreased. In summary, T106 might be a susceptible gene targeted by M. enterolobii. Exploration of such susceptible genes in plants is vital for finding new ways to control root-knot nematodes including M. enterolobii.

Brown rot, caused by Monilinia spp., is a serious threat to both stone fruit and pome fruit, greatly affecting the long-distance transportation and exportation of fruits. Based on genomic and transcriptomic analysis of infection of Monilinia fructicola on peach fruit, it was detected that the expression patterns of MfHMG5 and MfHMG6 genes in early stages of infection were similar, and both down-regulated significantly at 1 h after inoculation and then gradually increased. In order to investigate the biological functions of these two genes, the knockout and overexpression transformants of MfHMG5, and knockout and complemented transformants of MfHMG6 were obtained and the corresponding phenotypes were investigated. It was found that the knockout and overexpression of MfHMG5 gene decreased the growth rate and sporulation ability, but did not affect the pathogenicity of M. fructicola. Knock out of MfHMG6 gene reduced the growth rate, virulence and sporulation ability of M. fructicola, and led to the increased expression of MfHMG5 gene. These results indicated that HMG-box family genes MfHMG5 and MfHMG6 were involved in regulating the growth and pathogenesis of M. fructicola.

The occurrence of Fusarium crown rot (FCR) caused by Fusarium pseudograminearum has been becoming increasingly serious in China, which has posed a severe threat to wheat yield and quality. The SEY1 belongs to the RHD3 (Root Hair Defective 3) family and encodes a dynamin-like GTPase protein participating in endoplasmic reticulum (ER) fusion. The ER is involved in the synthesis of deoxynivalenol (DON) in different pathogenic fungi, while its function in F. pseudograminearum has not been reported. In this study, subcellular localization of GFP-tagged Sey1 (FpSey1) protein in F. pseudograminearum was observed, and the results showed that FpSey1 was localized in the ER. The FpSEY1 deletion mutant (ΔFpSey1) was generated through PEG-mediated protoplast transformation and verified by Southern blot analysis, and complemented strains were obtained as well. Compared with the wild-type strain, the ΔFpSey1 mutant exhibited significant reduction in vegetative growth, conidiation, relative expression of DON biosynthesis related genes (TRI1, TRI5, TRI10) , and the virulence on wheat coleoptiles and barley leaves. In addition, the ΔFpSey1 mutant is more sensitive to salt stress, hydrogen peroxide (H₂O₂), but more tolerant to dithiothreitol (DTT) than the wild-type and complemented strains. These results indicate that FpSey1 localized in the ER plays important roles in the growth and infection of F. pseudograminearum.

Leaf scald, caused by Xanthomonas albilineans (Xa), is a bacterial disease that seriously affects sugarcane production. Understanding the pathogenicity of this bacterial pathogen is crucial to preventing and controlling sugarcane leaf scald disease. Previous studies have shown that Phoq, a transmembrane histidine protein kinase in the two-component system of Xanthomonas, is a very important transduction factor, sensing extracellular signals, activating intracellular kinase activity, and subsequently regulating downstream gene expression. In this study, we collected Phoq protein sequences from eight pathogenic bacteria in the genus Xanthomonas, including Xa. The results of phylogenetic analysis and motif composition prediction showed that these Phoq proteins have conserved structure and similar physicochemical properties. To further investigate the biological role of phoq in Xa, we produced phoq knockout mutant in Xa-JG43 strain using the homologous recombination method. Compared with the wild-type strain Xa-JG43, the swimming ability and pathogenicity of the Xa-phoq knockout mutant were seriously weakened, but the swarming and stress response ability were not affected; In Xa-phoq complementary strain, the pathogenicity and swimming ability were restored to the level of the wild-type strain. This study provides a theoretical basis for further determination of the pathogenic mechanism of Xa.

Fusarium crown rot, mainly caused by Fusarium pseudograminearum, is a destructive disease in wheat production. To establish a rapid and reliable detection method for F. peasudeograminearum, the specific PCR primer pair (Fpg-F1/R2) was designed based on the RPB sequence, and real-time fluorescence quantitative PCR (qPCR) was used to validate the efficiency of the primer. The results showed that the primer pair had high specificity and sensitivity of 100 pg of DNA. Furthermore, the qPCR system for early and rapid detection of F. peasudeograminearum had an amplification efficiency of 87.5% and correlation coefficient of 0.99, and the pathologic threshold of F. pseudograminearum in soil was determined by using this detection system. It was found that F. pseudograminearum could cause Fusarium crown rot when the DNA concentration of F. pseudograminearum in field soil exceeded 213 pg·g-1. Hence, the qPCR-based method we developed for F. pseudograminearum detection has the advantages of high specificity and sensitivity, and can be used for rapid and early detection of F. pseudograminearum even in field soils.

Gray mold disease caused by Botrytis cinerea leads to severe crop yield reduction, and the secreted proteins play significant roles in the fungal infection. However, the functional mechanisms of these secreted proteins in B. cinerea remain largely unknown. In this study, a secreted protein, BcSGP1, from the secretome of B. cinerea during infection stages was identified. The expression level of BcSGP1 was upregulated during infection stages. Deletion of BcSGP1 caused reduction in pathogenicity, but not in growth rate, conidial production, or stress resistance. Transient expression of BcSGP1 in Nicotiana benthamiana leaves using agroinfiltration induced necrosis, and this necrosis-inducing activity depended on the plant receptor-like kinase BAK1, but not the SOBIR1. Furthermore, BcSGP1 could induce resistance against B. cinerea in N. benthamiana leaves. These results suggest that BcSGP1 is a pathogenesis-related secreted protein and involved in inducing plant resistance during the interaction between B. cinerea and plants. This study enhances our understanding of the pathogenic mechanisms of B. cinerea, providing a theoretical basis and genetic resources for effective control of gray mold disease.

In recent years, wheat scab, corn stalk rot and ear rot caused by Fusarium graminearum have led to substantial losses in crop yields. To investigate the genetic diversity and identify pathogenicity-related genes of F. graminearum, we performed population genetic diversity analysis and selective elimination analysis on 93 F. graminearum strains with released genome-wide resequencing data, using single nucleotide polymorphism (SNP) technology. The resequencing data of these F. graminearum strains were meticulously processed by using the Genome Analysis Toolkit 4 (GATK4), yielding a collection of 3,817,652 SNP markers. Based on these markers, a phylogenetic tree was constructed, and principal component analysis (PCA) and population structure analysis were conducted, effectively partitioning the 93 F. graminearum into 3 distinct groups. The selection elimination analysis of group 1 and group 2 revealed that group 1 exhibited a more pronounced response to selection pressure. A total of 70 regions were identified as candidate sites within the top 5% intersection region of population polymorphism (θπ) and population differentiation index (Fst). Furthermore, 76 protein-coding genes were identified in F. graminearum by leveraging the genomic location information. The results of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that these 76 candidate genes are mainly involved in metabolic pathways. Among them, eight genes (FGSG_05447, FGSG_05610, FGSG_10272, FGSG_10313, FGSG_01353, FGSG_05545, FGSG_10858 and FGSG_12745) are closely related to the pathogenicity of F. graminearum through further gene expression analysis. The result lays a basis for clarifying the pathogenic mechanism of F. graminearum and breeding F. graminearum-resistant wheat and maize varieties.

Late blight caused by Phytophthora infestans seriously affects the yield and quality of tomato. Previous research found that tomato plants contain age-related resistance (ARR) to P. infestans, but the underlying mechanisms are still unclear. Here, we used tomato variety ‘Micro Tom’ as the tested material and found that younger (4-week-old) plants are more resistant while older (8-week-old) plants are more susceptible to late blight. Through RNA sequencing and real-time quantitative PCR (qPCR) analysis, we observed that the transcription levels of genes involved in jasmonic acid (JA) synthesis, such as AOS1, AOS2 and AOC, are higher in 4-week-old plants than those in 8-week-old plants. We further examined the levels of several phytohormones and found that the concentration of JA in 4-week-old plants is significantly higher than that in 8-week-old plants. Transient expression of AOS1, AOS2 or AOC in tobacco leaves made them more resistant to late blight, suggesting that these JA biosynthetic genes can enhance tomato resistance to late blight. Tomato plants sprayed with MeJA were more resistant whereas tomato plants sprayed with JA synthesis inhibitor DIECA were more susceptible to late blight, suggesting that JA positively regulates tomato resistance to late blight. Thus, we provide evidence supporting a model in which genes involved in JA synthesis play important roles in the age-related resistance to late blight in tomato. Our results lay an important basis for using ARR to control tomato late blight.

PilZ domain-containing proteins are the largest known receptors of second messenger c-di-GMP in bacteria, but the functions and underlying mechanisms have not been reported in Pseudomonas syringae pv. actinidiae (Psa). To reveal the contribution and regulation mechanism of PilZ domain-containing proteins to the pathogenicity of Psa and to provide new ideas for controlling kiwifruit bacterial canker. Firstly, genome analysis and sequence alignment of Psa M228 were performed to identify the PilZ domain-containing proteins of Psa and analyze the conserved c-di-GMP binding motif. Then, homologous recombination was used to construct deletion mutants, and the pathogenicity, motility and growth between mutants and wild type were determined by leaf discs vacuum infiltration, soft agar plate assays and growth curve measurment, respectively. The transcripts of pathogenicity- and motility-relative genes in WT and ΔPsa_2195 mutant were measured by qRT-PCR. The results show that there are eight PilZ domain-containing proteins in Psa M228, among them PSA_2195 and PSA_1975 have neither a conserved RxxxR motif nor a (D/N)xSxxG binding motif that binds to c-di-GMP. The pathogenicity of ΔPsa_1116, ΔPsa_2195, ΔPsa_2203, ΔPsa_762, ΔPsa_4490 and ΔPsa_4763 were significantly reduced. Deletion of PSA_1116 and PSA_2195, PSA_3989, PSA_762 affected swimming motility and swarming motility, respectively. The growth curve of all mutants are no significant difference with wild type M228. Among all PilZ domain proteins, PSA_2195 regulates the transcription of flagella genes flgA, filE and T3SS genes. Taking together, our research revealed the function of eight PilZ domain-containing proteins in regulating pathogenicity and motility of Psa and the simple molecular mechanisms of PSA_2195.

After annotating the genome of Xanthomonas oryzae pv. oryzae strain PXO99A-GX, we found an atypical chemoreceptor gene PXO_01024, which encodes a protein with two transmembrane domains (TMD) and a methyl-accepting domain (MA) but without the ligand-binding domain (LBD). To understand the biological function of PXO_01024, we constructed the PXO_01024 deletion mutant DM01024 and its complemented strain CDM01024 by homologous double exchange method. Deletion of PXO_01024 resulted in the reduced formation of biofilm and almost loss of bacterial swimming, while in CDM01024 the swimming motility and biofilm formation ability were restored to wild-type levels. The virulence of DM01024 was not significantly different from that of the wild-type strain when inoculation of wounded host plants was performed. However, when unwounded plants were inoculated with the spraying inoculation method, the disease index caused by DM01024 was significantly reduced compared with that caused by the wild-type strain and complemented strain CDM01024, indicating that PXO_01024 played a role in early infection of Xoo. The chemotaxis of these strains was subsequently detected by capillary method, and the results showed that DM01024 showed significantly reduced chemotaxis to methionine, alanine, leucine, glycine, asparagine, phenylalanine, isoleucine, glucose, maltose, xylose, fructose, succinic acid, and tartaric acid compared with the wild-type strain. This study demonstrated that the atypical chemotactic receptor gene PXO_01024 is associated with chemotaxis, swimming motility, and early infection of Xoo.

Citrus bacterial canker caused by Xanthomonas citri subsp. citri (Xcci) is an important bacterial disea-se of citrus plants. Our previous work revealed that the homologous of XAC3126 (Accession no. AAM37971.1) of Xcci strain 306, which was predicted to be a single-domain response regulator, might be involved in the bacterial virulence in Xcci Guangxi wild-type strain N8. To investigate the biological functions of this gene (named embR), a deletion mutant was constructed with strain N8 as the starting strain. Simultaneously, a complemented strain was constructed using a recombinant plasmid harboring this innate gene. Phenotypic analysis revealed that the embR deletion mutant ΔembR showed obviously reduced virulence on the host plant Citrus reticulata Blanco 'Orah' compared with the wild-type strain, while the complemented strain exhibited similar virulence with that of the wild-type strain. Additionally, the ΔembR mutant displayed an obvious reduction in extracellular polysaccharide (EPS) production, cell motility and cell aggregation. Reverse transcription quantitative real-time PCR (RT-qPCR) revealed that the transcript level of a set of genes involved in EPS production and cell motility in the ΔembR mutant was decreased compared with that in the wild-type strain. These combined data indicate that the embR gene is required for multiple cellular processes including virulence in Xcci and modulates the expression of a series of virulence factor-related genes.

The nucleic acid sequence encoding the CI protein of potato virus Y (PVY) had multiple regions similar to prokaryotic promoter elements. It was possible to translate toxic proteins in prokaryotic cells, so it was difficult to construct vectors. According to the codon bias of prokaryotes, the CI sequence was modified without changing the amino acid sequence. The expression vector containing CI open reading frame (ORF) was successfully constructed, and CI protein was successfully expressed through the eukaryotic cell-free protein expression system. Six cell lines were prepared by immunizing Balb/c mice with purified protein using hybridoma technology. Indirect ELISA and western blot showed that the prepared CI monoclonal antibody 4B7_2D6(IgG1) had high sensitivity and specificity. The successful expression of CI protein provides a prerequisite for the purification of CI protein and the subsequent study of the structure and function of CI protein, which is of great significance for further exploring the interaction mechanism between CI and plant proteins.

Myb transcription factors play important roles in the regulation of various biological processes in plants. However, the molecular mechanism underlying their roles in regulating late blight resistance remains elusive. Here, we report the cloning of NbMybl, a Myb-like gene from Nicotiana benthamiana, which has an open reading frame of 753 bp and encodes a protein of 250 aa. NbMybl contains a Myb-like DNA-binding domain. Real-time quantitative PCR (qPCR) revealed that NbMybl was induced by infection with Phytophthora infestans. Subcellular localization analysis showed that NbMybl is located in both the nucleus and the cytoplasm. Silencing of NbMybl by virus-induced gene silencing (VIGS) significantly increased the susceptibility of plants to P. infestans. Transcriptome profiling by RNA sequencing identified 8468 differentially expressed genes (DEGs) with fold change ≥ 2 and FDR < 0.01 between NbMybl silenced and non-silenced control lines in response to P. infestans infection, and the result of RNA-seq was further validated by qPCR with 10 randomly selected DEGs. KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis revealed that a total of 373 DEGs were involved in plant-pathogen interaction, 308 DEGs and 216 DEGs were involved in MAPK signaling pathway and plant hormone signal transduction, respectively. We speculated that these DEGs might be closely related to the reduced resistance of NbMybl-silenced N. benthamiana lines to P. infestans. Our study provides valuable insights into the molecular mechanisms of NbMybl in regulating resistance to P. infestans.

Caspase is known to be involved in modulating apoptosis in mammals,controlling the occurrence and development of various diseases. In present study, the null mutant ΔFgCas4 and complemented strain ΔFgCas4-C of the caspase gene FgCas4 were obtained by gene disruption and complementation respectively in Fusarium graminearum. We observed that the FgCas4 deletion mutant (ΔFgCas4) did not affect the growth rate, colony morphology, conidiation, virulence and DON production. However, the deletion mutant ΔFgCas4 exhibited more hyphal branching and percentage of conidia with 3 septa increased by 8.7% compared to wild type PH-1 and complemented strain ΔFgCas4-C. External environmental stress assays showed that the gene disrupt mutant ΔFgCas4 became more sensitive to tested fungicides and metal ions. In addition, the lack of FgCas4 led to dramatically increased lipid droplet biosynthesis as well as increased resistance to osmotic stress agents. Subcellular localization showed that the caspase FgCas4 localized in vacuoles. On the other, the loss of FgCas4 resulted in earlier process of autophagy. Taken together, our study provides evidences that the caspase FgCas4 of Fusarium graminearum plays important roles in asexual reproduction, various environmental stress responses and auto-phagy regulation.

The bacterial wilt caused by Ralstonia solanacearum has brought a serious threat to tomato production. Coronatine (COR), structurally similar to JA-Ile (JA-isoleucine), is a compound produced by Pseudomonas syringae. In this study, we observed that pre-treatment of tomato seedlings with COR alleviated the symptoms caused by R. solanacearum. To investigate how COR affects tomato resistance to R. solanacearum, transcriptome sequencing of tomato seedlings inoculated with the pathogen 24 h after COR treatment was carried out. Analysis of RNA-seq data showed that COR treatment induced a total of 2122 differentially expressed genes (DEGs), including 998 up-regulated genes and 1124 down-regulated genes. DEGs annotation and pathway enrichment were conducted using GO database and KEGG database, and the results showed that COR affected the expression of genes related to plant-pathogen interaction pathway and plant hormone signaling pathways. Meanwhile, COR induced the up-regulation of genes involved in the jasmonic acid synthesis pathway and inhibited the expression of photosynthesis-related genes. Our results provide a theoretical basis for revealing the role of COR in plant-microbe interaction.