Rapid detection of latent infection by Puccinia striiformis f. sp. tritici (Pst) in wheat leaves is crucial for predicting and controlling wheat stripe rust, thereby safeguarding grain security. In this study, we used conventional PCR with the previously published primer combination betaf/betar to conduct molecular marker detection of Pst in wheat leaves at different inoculation times during the seedling stage. The results demonstrated that Pst amplification products were detectable 26 hours post-inoculation. Additionally, PCR amplification of 268 field-collected wheat leaf samples from six provinces (Shaanxi, Hubei, Anhui, Henan, Shandong, and Shanxi) revealed 45 samples (16.79%) potentially harboring latent Pst infections. Specifically, 29, 10, 3, 2, and 1 positive samples were detected in Henan, Shanxi, Shaanxi, Hubei, and Shandong provinces, respectively, while no latent Pst infections were identified in Anhui.

Pokkah boeng disease (PBD) is one of the most important fungal diseases threatening sugarcane production, and the major pathogen causing the disease is Fusarium sacchari in China. The survival factor Svf1 was first reported in Saccharomyces cerevisiae, which plays an important role in fungal growth, development and pathogenicity to plants. In this study, we found a Svf1 homologous gene FsSvf1 in the genome of F. sacchari, and the amino acid sequence similarity between the two proteins was 46.4%. The expression level of FsSvf1 was significantly up-regulated in sugarcane plant at 72 h post-inoculation with F. sacchari. Compared with the wild-type strain of F. sacchari, the FsSvf1 deletion mutant showed a significantly reduced mycelial growth rate, an obviously changed colony morphology on artificial media, and an increased sporulation ability; however, the number of perithecia produced by the FsSvf1 deletion mutant was reduced and the maturation time of ascus was delayed on carrot medium; under NaCl stress, the FsSvf1 deletion mutant showed increased sensitivity to NaCl, but under Congo red-, SDS- and H₂O₂-stressed conditions, the FsSvf1 deletion mutant exhibited reduced sensitivity to these stress agents; the pathogenicity of the FsSvf1 deletion mutant to sugarcane was also significantly reduced. These results demonstrate that FsSvf1 is a factor that plays important roles in the growth, development and pathogenicity of F. sacchari to plants, with the potential to be used as a target for disease control.

Nucleus-localized effector proteins secreted by plant pathogenic fungi can enter plant cell nucleus and interfere with host’s nuclear biological functions. It is of great significance to elucidate the underlying molecular mechanism of nucleus-localized effector proteins in regulating host immune response during Gymnosporangium yamadae-Malus domestica interaction. In this study, the subcellular localization of the two candidate nucleus-localized effector proteins from G. yamadae, GyBarwin55 and GyRlpA22 obtained in our previous study, was determined via Agrobacterium tumefaciens-mediated transient expression in Nicotiana benthamiana, and the result showed that GyBarwin55 was specifically localized to the nucleus, and GyRlpA22 was localized to both cytoplasm and nucleus. Quantitative real-time PCR (qPCR) analysis result showed that the expression level of these two effector protein-coding genes in apple seedlings was significantly up-regulated during the spermatial development stage of G. yamadae. The results of heterologous expression in Nicotiana benthamiana mediated by A. tumefaciens, combined with that of in situ expression in apple leaves, demonstrated that both GyBarwin55 and GyRlpA22 have elicitor activities. However, site-directed mutagenesis of the nuclear localization sequence (NLS) resulted in the loss of the ability of GyBarwin55 and GyRlpA22 to localize to the nucleus and also to act as elicitors. The above results revealed that the NLS sequence is indispensable for both GyBarwin55 and GyRlpA22 to localize to nucleus and function as elicitors, and these two nucleus-localized effector proteins play important roles during the interaction between G. yamadae and apple tree.

Peach brown rot disease caused by Monilinia fructicola has resulted in huge economic losses to peach production in China, and identification of the key pathogenic factors of M. fructicola is crucial for targeted prevention and control of the disease it caused. To investigate the biological functions of MfHOX1 in M. fructicola, the Split-Marker method and PEG-mediated homologous recombination transformation were used here to obtain the MfHOX1 knockout mutant and its complementary strain. Compared with the wild-type strain of M. fructicola, the mycelial growth rate of the MfHOX1 knockout mutant was significantly decreased, while the conidial production was increased; the sensitivities of MfHOX1 knockout mutant to exogenous stresses including H₂O₂, SDS, CR, CaCl₂ and NaCl were significantly decreased, but those to glycerol, sorbitol and glucose were increased. In addition, the virulence of the MfHOX1 knockout mutant was significantly reduced, and the expression levels of several pathogenicity-related genes such as MfAP1, MfPG1, MfCUT1, MfPmk1, MfSSP and MfHsbA1 were significantly increased due to the deletion of the MfHOX1 gene. The aforementioned results suggest that MfHOX1 plays roles in regulating mycelial growth, sporulation, pathogenesis and external stress responses.

Rubber tree powdery mildew pathogen Oidium heveae, complete its infection cycle on living host plant through secreting a plethora of effectors proteins. The expression of candidate effector gene CSEP03399, encoding an N terminal secretion signal peptide, was induced during O. heveae infection on Arabidopsis. No obvious cell deaths were observed after CSEP03399 expression in transgenic Arabidopsis plants and Nicotiana benthamiana, suggesting that CSEP03399 is not an avirulence effector protein. After inoculated with O. heveae, CSEP03399 can obviously enhance the fungal growth in early stage and the conidospores formation in late stage, The ROS production, cell death and the expression of PR1(pathogenesis related 1)gene were significantly reduced. All this indicated that CSEP03399 was a virulence effector protein for plant cell. However, the hypersensitive responses triggered by powdery mildew resistance gene WRR4B (white rust resistance 4B), Pseudomonas syringae DC 3000 avrB and DC 3000 avrRpt2 were not inhibited by CSEP03399. Nonetheless, the expressions of chitin-induced FRK1 (FLG22-induced receptor-like kinase 1) and WRKY22 gene were suppressed by CSEP03399 and the pathogenicity of P. syringae DC 3000 and DC 3000 hrcC^- was enhanced after Arabidopsis treated with chitin. Overall, these results suggested that CSEP03399 may fulfill its virulence by inhibiting the chitin-triggered immune signaling.

In the study, a metacaspases gene was islated from the cDNA library of the interaction between the wheat cultivar ‘Suwon 11’ and Puccinia striiformis West. f. sp. tritic Eriks.＆ Henn.(Pst). The gene was designed as TaMCA3. Through transient overexpression experiments, it was found that the TaMCA3 gene can effectively inhibit cell necrosis induced by the cell necrosis inducer BAX. The protoplast subcellular localization experiment showed that the TaMCA3 protein is mainly distributed in the cytoplasm. During the compatible interaction between wheat and Pst, it is shown that TaMCA3 participates in the susceptibility process of wheat to Pst. After using VIGS (virus-induced gene silence) technology to transiently silence TaMCA3, in comparison to the control plants, the silenced plants exhibited obvious necrotic spots on their leaves, and the number of urediniospore pustules of Pst CYR31 was significantly reduced. This indicates that silencing TaMCA3 can significantly enhance wheat′s resistance to Pst CYR31. Furthermore, by using yeast two-hybrid screening identified TaASP, a left-handed aspartate protease, was identified as the interacting target of TaMCA3. In vitro and in vito protein interaction experiments confirmed the interaction between TaASP and TaMCA3. This discovery provides new insights for a deeper understanding of the specific functions and mechanisms of TaMCA3 in the interaction between wheat and Pst.

In this study, hyphal growth rate and spore production methods were used to determine the indoor inhibitory effect of difenoconazole on Coryneum populinum. Morphotoxicology and transcriptome sequencing were used to analyze the treatment of Differentially expressed genes (DEGs) after treatment with difenoconazole for exploring the antibacterial mechanism of difenoconazole. The results showed that the higher the concentration of difenoconazole treatment, the greater the inhibition effect on C. populinum with lower rates of colony growth and sporulation. After treatment with difenoconazole, 618 DEGs were obtained from C. populinum. GO enrichment analysis showed that DEGs were the most abundant in cell anatomical entities, essential components of membranes, intrinsic components of membranes, and carbohydrate metabolism. KEGG enrichment analysis showed that alanine, aspartic acid and glutamate metabolism, MAPK (mitogen-activated protein kinase) signaling, ribosomal biosynthesis and metabolism were all in the first 20 enrichment pathways. Twenty-six key genes were up-regulated and nine key genes were down-regulated in MAPK signaling, including Ste2, Cdc42, Ste18 and Mcm1 in the Fus3/Kss1 signaling pathway (P<0.05), Cdc42 in the HOG signaling pathway (P<0.05), and CWI signaling pathways Wsc1, Slt2 and Fks2 in the up-regulated manners (P<0.05). The results provided data support for revealing the response mechanism of C. populinum to difenoconazole stress and research basis for the further development of synergistic agents of triazole fungicides.

The monopartite geminiviral V2 protein plays an important role in the viral infection process. Howe-ver, there are few studies on the function of the AV2 protein of bipartite geminiviruses. This study investigated the interaction between the bipartite tomato leaf curl Hsinchu virus (ToLCHsV) AV2 protein and the Nicotiana benthamiana Ran binding protein 1-2b (NbRanBP1-2b), and analyzed their effect on the pathogenicity of the virus. Bimolecular fluorescence complementation (BiFC), GST pull-down and subcellular co-localization were used to validate the interaction between ToLCHsV AV2 and NbRanBP1-2b. NbRanBP1-2b was observed to localize at both the cell membrane and cytoplasm. When ToLCHsV AV2 co-localized with NbRanBP1-2b, the original localization of AV2 and NbRanBP1-2b was altered. After inoculated with ToLCHsV, N. benthamiana leaves exhibited wrinkling, accompanied by delayed growth and development. Furthermore, the relative expression level of NbRanBP1-2b exhibited a gradual increase. When NbRanBP1-2b was silenced by tobacco rattle virus (TRV) or overexpressed by potato virus X (PVX), the virus accumulation in the inoculated N. bentha-miana plants was relatively decreased. These results indicate that ToLCHsV can effectively utilize NbRanBP1-2b to promote virus infection by interacting with its AV2 protein during infection.

Fusarium wilt of banana, caused by Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4), is a devastating soil-borne disease threatening global banana production. The conidia and chlamydospores in the soil are the primary inoculum for this disease. The C2H2-type zinc-finger transcription factor FlbC in Aspergillus nidulans is a key regulator of conidial development. In this study, the FlbC homolog gene FocFlbC was identified in Foc TR4, and its knockout mutant (ΔFocFlbC) and complemented strain were constructed using protoplast-mediated genetic transformation technology. The subcellular localization and biological functions of this protein were analyzed. The results showed that the FocFlbC protein was localized to the nucleus in both hyphae and conidia. Compared to the wild-type (WT) strain, the ΔFocFlbC mutant exhibited significantly reduced mycelial growth rate on maltose medium, while growth on other carbon sources showed no significant difference; conidiation of the ΔFocFlbC mutant was significantly reduced on all tested carbon sources. Furthermore, the ratio of conidiation between the WT and mutant was highest on maltose medium, differing significantly from other carbon sources. Although the FocFlbC deletion mutation showed no significant effect on biomass accumulation or conidial germination, the mutant exhibited the following phenotypic defects compared to the wild-type strain: reduced tolerance to cell wall and salt stresses; decreased enzymatic activities of α-amylase, filter paper cellulase, and β-1,4-D-glucanase, accompanied by downregulated expression of corresponding hydrolase genes; significantly reduced virulence. These phenotypic defects were restored in the complemented strain. In conclusion, FocFlbC not only regulates hyphal growth on maltose and conidial development of Foc TR4, but also participates in regulating cell wall integrity, salt stress response, hydrolase synthesis, and virulence. The results provide a theoretical basis for further elucidating the molecular mechanism underlying the growth, development, and pathogenicity of Foc TR4.

Rice dwarf disease, caused by rice dwarf virus (RDV), poses a significant threat to rice production. Establishing a RDV monitoring system is critical for early field surveillance and disease control. In this study, a 1 056 bp fragment of the P9 gene of RDV was amplified via reverse transcription-PCR (RT-PCR) from RDV-infected rice plant and expressed in Escherichia coli BL21 (DE3). Purified P9 protein was used to immunize New Zealand white rabbits, generating P9 polyclonal antiserum. Enzyme-linked immunosorbent assay (ELISA) revealed an antiserum titer of 1∶32 000. Western blot analysis confirmed that the antigen-affinity-purified polyclonal antibody specifically recognized RDV P9 protein in both infected rice plants and viruliferous insect vectors. The antibody was further applied in indirect ELISA and dot-ELISA assays, combined with RT-PCR, to analyze 223 field samples (rice, barnyard grass, and three insect vectors: Nephotettix cincticeps, Recilia dorsalis, and Nilaparvata lugens) collected from Xianyou County, Putian City, Fujian Province. The results showed RDV infection rates of 29.03% in rice and 19.05% in barnyard grass, with viral carrier rates of 12.28% in N. cincticeps, 2.56% in R. dorsalis, and 0 in N. lugens. The RDV P9 polyclonal antibody exhibited high titer and specificity. This study successfully developed high-titer, high-specificity polyclonal antibodies against RDV P9. The established serological methods not only enhance diagnostic reliability for rice dwarf disease but also provide critical technical support for future investigations into P9 protein function and field-based RDV detection.

High-throughput sequencing has become a crucial tool for advancing our understanding of fungal genomics and transcriptomics. However, accurately assessing gene expression levels from RNA sequencing (RNA-Seq) data necessitates the precise mapping of short sequence reads back to their correct locations in the reference genome. Sequenced strains often exhibit sequence variations from the reference genome strains, and due to the reference genome containing only one type of nucleotide information at any given locus, reads carrying alternative nucleotide information will have at least one mismatch with the reference genome, leading to mapping biases during the read alignment. To address this bias, researchers have developed various software and algorithms. Nevertheless, the applicability of these methods for fungal RNA-Seq data remains unclear. In this study, we took Fusarium graminearum, an important plant pathogenic fungus, as an example. Utilizing both simulated and real RNA-Seq data of this fungus, we comprehensively assessed the mapping biases and mapping rates of various mapping methods when dealing with single nucleotide polymorphisms (SNPs), RNA editing, and sequencing errors in reads. The results indicate that the graph-based genomic approach using the vg software outperforms others, demonstrating optimal performance. This method requires lower sequencing depth and significantly reduces mapping biases caused by polymorphic sites while improving reads mapping rates. These findings provide a reference for the analysis of complex fungal RNA-Seq data.

Carya illinoensis is an important non-timber forest species in China, and one of the most serious diseases caused by Colletotrichum spp. dangerously threaten the development of the pecan industry. In order to investigate the pathological mechanism of Colletotrichum spp. in pecan, C. fructicola B-5 was used to establish and applied the genetic transformation system. The results showed that mycelium was cultured in CM medium for 20 h, 1% lysing enzyme was lysed at 30 °C for 3 h. The production of protoplasts was up to 8.92×10⁶ cells·mL^-1. Using PEG-mediated protoplast transformation, the plasmid with the resistance gene and GFP gene can be transferred into B-5 protoplasts, and the biological characteristics of the positive transformant B-5(GFP) did not change significantly. The B-5(GFP) strain was used to analyze the infection dynamics of Colletotrichum spp. on the host leaves. The results showed that the conidia began to germinate on the surface of the host after 9 h, began to infect the host mesophyll tissue after 48 h, and a large number of primary hyphae were produced after 96 h, and the infected part showed cell death. The genetic transformation system of C. fructicola in pecan was successfully established in this study, and the infection dynamics of C. fructicola B-5(GFP) on pecan were preliminarily clarified, which laid a foundation for subsequent pathogenic mechanism analysis and prevention and control.

In this study, an infectious clone of zucchini yellow mosaic virus (ZYMV) was constructed, and the differentially expressed genes (DEGs) of ZYMV-infected Cucumis melon were analyzed via RNA-Seq technology. The RNA was extracted from the leaves of ZYMV-infected C. melon, the 3 segments divided from the full genome of CGMMV were cloned separately by using RT-PCR, and then the ZYMV infectious clone was constructed through homologous recombination. Healthy C. melon was inoculated with pCB-ZYMV, typical mosaic symptoms were observed in the C. melon at 7 days post inoculation (dpi), and ZYMV CP protein could be detected by Western blot. The total RNAs extracted from the ZYMV-infected and healthy C. melon leaves, respectively, were sent to the biotech company for high-throughput transcriptome sequencing analysis. A total of 3 108 differentially expressed genes (DEGs) were screened, of which 1 558 were up-regulated and 1 550 were down-regulated. GO, COG annotation and KEGG pathway analysis revealed the involvement of many DEGs in host plant carbohydrate transport and metabolism, plant signal transduction, plant-pathogen interactions, starch and sucrose metabolism, and MAPK cascade response and other physiological metabolic pathways related to disease resistance. To validate the RNA-seq results, the differential expression of eight genes were examined by qRT-PCR, which was basically consistent with the transcriptome results. This study is of great significance for elucidating the molecular mechanism of ZYMV resistance in melon and breeding selection of ZYMV resistant melon varieties.

Bioinformatics methods were used in this study to explore the key ZjDELLA genes that are responsive to JWB (Jujube witches′ broom) phytoplasma infection in jujube plants based on the ‘Junzao’ genome information. Forty-three ZjGRAS TFs (ZjGRAS1-ZjGRAS43), which were divided into 9 subfamilies, were identified, and 4 of them were identified as jujube DELLA subfamily (ZjDELLA) TFs. According to the functional prediction network of ZjDELLA we constructed, jujube DELLA protein might play a role in GA signal transduction pathway. The expression of ZjDELLA1, ZjDELLA2 and ZjDELLA4 were significantly up-regulated, while ZjDELLA3 was significantly down-regulated in response to JWB phytoplasma infection. Additionally, heterologous expression of ZjDELLA4 in Arabidopsis could induce leaflet and dwarf phenotypes. To further investigate the mechanism underlying ZjDELLA4-inducing dwarfism in Arabidopsis, quantitative real-time PCR (qPCR) analysis was performed to determine the expression patterns of GA signal transduction-related genes. It was found that ZjDELLA4 significantly up-regulated the expression of GID1A, GID1B and GID1C, while significantly suppressed the expression of SPY. The results indicate that ZjDELLA4 may induce abnormal growth phenotypes in Arabidopsis by interfering with GA signal transduction.

The effects of synergism between sweet potato chlorotic stunt virus (SPCSV) and sweet potato feathery mottle virus (SPFMV) on photosynthesis, submicroscopic leaf epidermis structure, and chloroplast ultrastructure of sweet potato were studied using sweet potato plants infected with either SPCSV, SPFMV, or a combination of the two. The results showed that the net photosynthetic rate, stomatal conductance, transpiration rate, CO₂ utilization efficiency, and chlorophyll content of sweet potato plants were significantly decreased, and the activities of antioxidant enzymes and malondialdehyde content were increased due to the co-infection of SPCSV and SPFMV. Scanning electron microscopy showed that the co-infection of SPCSV and SPFMV could induce stomatal closure, leaf surface depression, and guard cell shrinkage, whereas no significant changes occurred in the single-infected plants,compared to the healthy plants. By using a transmission electron microscope, we found that the co-infection with SPCSV and SPFMV resulted in a reduction in the number and severe distortion of chloroplasts and the presence of large numbers of cylindrical inclusions in the cytoplasm of sweet potato plants. In the single-infected sweet potato plants, the number of chloroplasts remained unchanged, while some alternations on the ultrastructure were found, including increasedswollen starch granules and some loosely arranged grana and stroma lamellae.

Botrytis cinerea, a phytopathogenic fungi with a wide host range, can cause gray mold disease in many important crops. During infection of plant, B. cinerea secretes numerous cell death-inducing proteins (CDIPs) to induce host cell death, which as a result promotes its infection. In this study, we analyzed the secreted proteome during the infection stage of B. cinerea and identified a secreted protein BcXYG3, which contains GH12 and fCBD domains. Transiently expression of BcXYG3 rather than BcXYG3^Δsp (BcXYG3 without a signal peptide) in Nicotiana benthamiana leaves could induce cell death, suggesting that BcXYG3 possibly functions in plant cell apoplastic space. The expression of BcXYG3 was upregulated during the infection stage of B. cinerea. However, deletion or over-expression of BcXYG3 did not significantly affect the pathogenicity, growth rate, conidial production, and some stress tolerance of the pathogen. In addition, infiltration of purified BcXYG3 into N. benthamiana leaves could trigger plant resistance and the expression of defense-related genes. In conclusion, the secreted protein BcXYG3 of B. cinerea can trigger cell necrosis and resistance in plant, playing significant roles in the interaction between B. cinerea and host plant. The result is helpful for clarifying the mechanism underlying B. cinerea-plant interaction and provides theoretical basis and genetic resource for breeding of resistant crop varieties against gray mold disease.

Peach brown rot is one of the major diseases of peach, which causes significant losses to growers when it occurs in large scale. Currently, research on brown rot disease mainly focuses on the isolation and identification of the pathogen, fungicides resistance, and control measures. While the study on the pathogenic mechanism of the causing agent Monilinia fructicola, is relatively rare. This study is based on the previously obtained genome of M. fructicola and its early-stage transcriptome from infected fruit. Through PHI annotation analysis, it was found that the MfATG1 gene may play an important role in the infection process of M. fructicola. By using gene knockout technology, the knockdown positive transformants of MfATG1 gene were obtained, and their growth and development, virulence, and other stress-resistant phenotypes were compared with the wild-type strain to analyze the biological functions of the gene. The research results showed that the knockdown of MfATG1 gene affected the mycelial morphology and autophagy process, reduced mycelial growth rate and virulence of M. fructicola, but does not affect spore germination, indicating that MfATG1 gene is involved in the regulation of mycelial growth, virulence, and autophagy process in M. fructicola. Under NaCl stress conditions, the knockdown transformants increased the sensitivity to NaCl, indicating that the MfATG1 gene is involved in the response to salt stress. Under H₂O₂, SDS, and Congo red stress conditions, the knockdown transformants reduced the sensitivity to them, indicating that MfATG1 gene participates in the control of cell wall integrity and the oxidative stress response in M. fructicola.

The nuclear transcription factor Y (NF-Y) plays an important role in plant growth and development and in the response to abiotic stresses, but the mechanisms by which they are involved in insect and microbial interactions are less well understood. The purpose of this study was to identify the gene members of the NF-Y family of sorghum by bioinformatics, analyze their sequence characteristics, determine the spatio-temporal differences in gene expression in sorghum tissues, respond to Melanaphis sacchari and Sporisorium reilianum infection and DNA variation analysis. In the sorghum genome screened to 9 NF-YA subfamilies, 15 NF-YB subfamilies and 12 NF-YC subfamilies, is unevenly distributed on 10 chromosomes, and the gene structure of members in the same subfamily is similar, and most of the closely related members of the SbNF-Y family have similar exon-intron structures and conservative motifs. The encoded NF-Y proteins are all hydrophilic proteins, and the secondary structure is dominated by α helix and random curl; The results of protein domain analysis showed that the CBFB_NFYA was a conserved domain of the NF-YA subfamily, CBFD_NFYB_HMF a conserved domain of the NF-YB and NF-YC subfamilies. Analysis of gene promoter cis-acting elements showed that the promoter region of the NF-Y family of sorghum contained a large number of core elements, such as photoresponsive elements and a variety of elements that responded to the environment and hormones. Phylogenetic tree analysis showed that the NF-Y family was relatively conserved in the evolutionary process, and there was no obvious differentiation between monocotyledons and dicotyledons, and the relationship between sorghum NF-Y and maize was the closest; Gene expression analysis showed that there were significant differences in the expression of members of the NF-Y family of sorghum in different sorghum varieties and tissues. Expression of SbNF-YB3.2g and SbNF-YB11.7g were significantly enhanced after cane aphid infestation. Expression of SbNF-YB10.2g and SbNF-YB11.7g were significantly enhanced by head smut fungi infection. SNP/INDEL DNA variations were identified for future development of breeding selection. This study preliminarily analyzed the gene structure and expression of the NF-Y family of sorghum, aiming to provide a theoretical basis for the breeding of sorghum aphid resistance and head smut resistance.

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.