Extracellular perception of multiple novel core effectors from the broad host-range pear anthracnose pathogen Colletotrichum fructicola in the nonhost Nicotiana benthamiana.

Colletotrichum fructicola is emerging as a devastating pathogenic fungus causing anthracnose in a wide range of horticultural crops, particularly fruits. Exploitation of nonhost resistance (NHR) represents a robust strategy for plant disease management. Perception of core effectors from phytopathogens frequently leads to hypersensitive cell death and resistance in nonhost plants; however, such core effectors in C. fructicola and their signaling components in non-hosts remain elusive. Here, we found a virulent C. fructicola strain isolated from pear exhibits non-adaptation in the model plant Nicotiana benthamiana. Perception of secreted molecules from C. fructicola appears to be a dominant factor in NHR, and four novel core effectors-CfCE4, CfCE25, CfCE61, and CfCE66-detected by N. benthamiana were, accordingly, identified. These core effectors exhibit cell death-inducing activity in N. benthamiana and accumulate in the apoplast. With a series of CRISPR/Cas9-edited mutants or gene-silenced plants, we found the coreceptor BAK1 and helper NLRs including ADR1, NRG1, and NRCs mediate perceptions of these core effectors in N. benthamiana. Concurrently, multiple N. benthamiana genes encoding cell surface immune receptors and intracellular immune receptors were greatly induced by C. fructicola. This work represents the first characterization of the repertoire of C. fructicola core effectors responsible for NHR. Significantly, the novel core effectors and their signaling components unveiled in this study offered insights into a continuum of layered immunity during NHR and will be helpful for anthracnose disease management in diverse horticultural crops.

Ustilaginoidea virens-secreted effector Uv1809 suppresses rice immunity by enhancing OsSRT2-mediated histone deacetylation.

Rice false smut caused by Ustilaginoidea virens is a devastating rice (Oryza sativa) disease worldwide. However, the molecular mechanisms underlying U. virens-rice interactions are largely unknown. In this study, we identified a secreted protein, Uv1809, as a key virulence factor. Heterologous expression of Uv1809 in rice enhanced susceptibility to rice false smut and bacterial blight. Host-induced gene silencing of Uv1809 in rice enhanced resistance to U. virens, suggesting that Uv1809 inhibits rice immunity and promotes infection by U. virens. Uv1809 suppresses rice immunity by targeting and enhancing rice histone deacetylase OsSRT2-mediated histone deacetylation, thereby reducing H4K5ac and H4K8ac levels and interfering with the transcriptional activation of defence genes. CRISPR-Cas9 edited ossrt2 mutants showed no adverse effects in terms of growth and yield but displayed broad-spectrum resistance to rice pathogens, revealing a potentially valuable genetic resource for breeding disease resistance. Our study provides insight into defence mechanisms against plant pathogens that inactivate plant immunity at the epigenetic level.

Post-Translational Modification ß-Hydroxybutyrylation Regulates Ustilaginoidea virens Virulence.

Lysine ß-hydroxybutyrylation (Kbhb) is an evolutionarily conserved and widespread post-translational modification that is associated with active gene transcription and cellular proliferation. However, its role in phytopathogenic fungi remains unknown. Here, we characterized Kbhb in the rice false smut fungus Ustilaginoidea virens. We identified 2204 Kbhb sites in 852 proteins, which are involved in diverse biological processes. The mitogen-activated protein kinase UvSlt2 is a Kbhb protein, and a strain harboring a point mutation at K72, the Kbhb site of this protein, had decreased UvSlt2 activity and reduced fungal virulence. Molecular dynamic simulations revealed that K72bhb increases the hydrophobic solvent-accessible surface area of UvSlt2, thereby affecting its binding to its substrates. The mutation of K298bhb in the septin UvCdc10 resulted in reduced virulence and altered the subcellular localization of this protein. Moreover, we confirmed that the NAD+-dependent histone deacetylases UvSirt2 and UvSirt5 are the major enzymes that remove Kbhb in U. virens. Collectively, our findings identify regulatory elements of the Kbhb pathway and reveal important roles for Kbhb in regulating protein localization and enzymatic activity. These findings provide insight into the regulation of virulence in phytopathogenic fungi via post-translational modifications.

Nitrate reductase is required for sclerotial development and virulence of Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum, the causal agent of Sclerotinia stem rot (SSR) on more than 450 plant species, is a notorious fungal pathogen. Nitrate reductase (NR) is required for nitrate assimilation that mediates the reduction of nitrate to nitrite and is the major enzymatic source for NO production in fungi. To explore the possible effects of nitrate reductase SsNR on the development, stress response, and virulence of S. sclerotiorum, RNA interference (RNAi) of SsNR was performed. The results showed that SsNR-silenced mutants showed abnormity in mycelia growth, sclerotia formation, infection cushion formation, reduced virulence on rapeseed and soybean with decreased oxalic acid production. Furthermore SsNR-silenced mutants are more sensitive to abiotic stresses such as Congo Red, SDS, H2O2, and NaCl. Importantly, the expression levels of pathogenicity-related genes SsGgt1, SsSac1, and SsSmk3 are down-regulated in SsNR-silenced mutants, while SsCyp is up-regulated. In summary, phenotypic changes in the gene silenced mutants indicate that SsNR plays important roles in the mycelia growth, sclerotia development, stress response and fungal virulence of S. sclerotiorum.

Cytochrome bc1 Complex: Potential Breach to Improve the Activity of Phenazines on Xanthomonas.

The effects of the natural pesticides, phenazines, were reported to be limited by some tolerant metabolism processes within Xanthomonas. Our previous studies suggested that the functional cytochrome bc1 complex, the indispensable component of the respiration chain, might participate in tolerating phenazines in Xanthomonas. In this study, the cytochrome bc1 mutants of Xanthomonas campestris pv. campestris (Xcc) and Xanthomonas oryzae pv. oryzae (Xoo), which exhibit different tolerance abilities to phenazines, were constructed, and the cytochrome bc1 complex was proven to partake a critical and conserved role in tolerating phenazines in Xanthomonas. In addition, results of the cytochrome c mutants suggested the different functions of the various cytochrome c proteins in Xanthomonas and that the electron channeled by the cytochrome bc1 complex to cytochrome C4 is the key to reveal the tolerance mechanism. In conclusion, the study of the cytochrome bc1 complex provides a potential strategy to improve the activity of phenazines against Xanthomonas.

Effect of different straw retention techniques on soil microbial community structure in wheat-maize rotation system.

Rotational straw return technique is considered an effective measure for improving soil quality and maintaining soil microorganisms. However, there are few reports on the influence of wheat-maize crop rotation and straw-returning tillage on crop soil microbial communities in China. This study aimed to investigate how wheat or maize straw-incorporation practices affect bacterial and fungal communities under wheat-maize rotational farming practices. To clarify the effects of straw incorporation on microbial composition, microbial communities from soils subjected to different treatments were identified using high-throughput sequencing. Our results showed that, before corn planting, wheat and maize straw returning reduced bacterial density and increased their diversity but had no effect on fungal diversity. However, before wheat planting, returning wheat and corn stalks to the field increased the diversity of soil bacteria and fungi, whereas returning corn stalks to the field reduced the diversity of fungi and other microorganisms. Straw return significantly increased the relative abundance of Ascomycota in the first season and decreased it in the second season; however, in the second season, wheat straw return increased the relative abundance of Bradyrhizobium, which can promote the soil microbial nitrogen cycle and provide nitrogen to the soil. Wheat and maize straw return increased the relative abundance of Chaetomium, whereas, individually, they decreased the relative abundance. In addition, we detected two fungal pathogens (Fusarium and Trichoderma) under the two planting patterns and found that the relative abundance of pathogenic Fusarium increased with wheat straw return (FW and SW). Trichoderma increased after treatment with maize straw return before wheat planting (S group). These results suggest that wheat straw return (FW and SW) and maize straw return might have a negative impact on the pathogenic risk. Therefore, further studies are needed to determine how to manage straw returns in agricultural production.

MoNap1, a Nucleosome Assemble Protein 1, Regulates Growth, Development, and Pathogenicity in Magnaporthe oryzae.

Nap1 is an evolutionarily conserved protein from yeast to human and is involved in diverse physiological processes, such as nucleosome assembly, histone shuttling between the nucleus and cytoplasm, transcriptional regulation, and the cell cycle regulation. In this paper, we identified nucleosome assemble protein MoNap1 in Magnaporthe oryzae and investigated its function in pathogenicity. Deletion of MoNAP1 resulted in reduced growth and conidiation, decreased appressorium formation rate, and impaired virulence. MoNap1 affects appressorium turgor and utilization of glycogen and lipid droplets. In addition, MoNap1 is involved in the regulation of cell wall, oxidation, and hyperosmotic stress. The subcellular localization experiments showed that MoNap1 is located in the cytoplasm. MoNap1 interacts with MoNbp2, MoClb3, and MoClb1 in M. oryzae. Moreover, deletion of MoNBP2 and MoCLB3 has no effects on vegetative growth, conidiation, and pathogenicity. Transcriptome analysis reveals that MoNAP1 is involved in regulating pathogenicity, the melanin biosynthetic process. Taken together, our results showed that MoNap1 plays a crucial role in growth, conidiation, and pathogenicity of M. oryzae.

Development of Recombinase Polymerase Amplification Combined with Lateral Flow Detection Assay for Rapid and Visual Detection of Ralstonia solanacearum in Tobacco.

Bacterial wilt caused by Ralstonia solanacearum is a serious soilborne disease that results in severe losses to tobacco (Nicotiana tabacum) production in China. In this study, a novel RPA-LFD assay for the rapid visual detection of R. solanacearum was established using recombinase polymerase amplification (RPA) and lateral-flow dipstick (LFD). The RPA-LFD assay was performed at 37°C in 30 min without complex equipment. Targeting the sequence of the RipTALI-9 gene, we designed RPA primers (Rs-rpa-F/R) and an LF probe (Rs-LF-probe) that showed high specificity to R. solanacearum. The sensitivity of RPA-LFD assay to R. solanacearum was the same as that in conventional PCR at 1 pg genomic DNA, 103 CFU/g artificially inoculated tobacco stems, and 104 CFU/g artificially inoculated soil. The RPA-LFD assay could also detect R. solanacearum from plant and soil samples collected from naturally infested tobacco fields. These results suggest that the RPA-LFD assay developed in this study is a rapid, accurate molecular diagnostic tool with high sensitivity for the detection of R. solanacearum.

The RPA190-pc gene participates in the regulation of metalaxyl sensitivity, pathogenicity and growth in Phytophthora capsici.

Metalaxyl is one of the main fungicides used to control pepper blight caused by Phytophthora capsici. Metalaxyl resistance of P. capsici, caused by the long-term intense use of this fungicide, has become one of the most serious challenges facing pest management. In this study, a conserved domain RPOLA-N of the RPA190 gene of P. capsici (RPA190-pc) was identified from the P. capsici SD1-9 strain. The role of the RPA190-pc underlying the metalaxyl resistance of P. capsici was investigated. Three P. capsici mutants, two with downregulated RPA190-pc (SD1-9C-3 and C-4) expression and one showing upregulation (OESD1-9-1), were obtained by Polyethylene Glycol (PEG) mediated protoplast transformations of P. capsici SD1-9. Quantitative real-time reverse transcription PCR results showed that RPA190-pc was downregulated by more than 60% in SD1-9C-3/C-4 and upregulated 3-fold in OESD1-9-1 compared with that of the control strain SD1-9. Evaluation of the metalaxyl resistance of these three transformants showed that the EC50 values of metalaxyl against SD1-9C-3, SD1-9C-4, and OESD1-9-1 were 120.0 µg·mL-1, 24.4 µg·mL-1, and 15573.0 µg·mL-1, respectively, corresponding to 63.3% decrease, 92.5% decrease, and 47.7-fold increase relative to the EC50 value in SD1-9. Compared with SD1-9, the mycelia of transformants SD1-9C-3, SD1-9C-4, and OESD1-9-1 showed more branches and shorter branches; and the transformants had different pathogenicity to different hosts plants. The expression of the candidate gene RPA190-pc during 10 life-history stages was further studied, the results showed that expression level reached a maximum at the zoospores stage, and it gradually increased with the increase of SD1 and SD1-9 infection time of pepper leaves, indicated that RPA190-pc may be related to the growth and pathogenicity of P. capsici. These results indicate that the expression of RPA190-pc is involved in the regulation of P. capsici resistance to metalaxyl.

Proteomics Reveals the Mechanism Underlying the Inhibition of Phytophthora sojae by Propyl Gallate.

Phytophthora sojae is a serious soil-borne pathogen, and the major control measures undertaken include the induction of soybean-resistance genes, fungicides, and scientific and reasonable planting management. Owing to the safety and resistance of fungicides, it is of great importance to screen new control alternatives. In a preliminary study, we observed that propyl gallate (PG) exerts a considerable inhibitory effect on P. sojae and can effectively prevent and cure soybean diseases, although the underlying mechanism remains unclear. To explore the inhibitory mechanism of PG on P. sojae, we analyzed the differences in the protein profile of P. sojae before and after treatment with PG using tandem mass tag (TMT) proteomics. Proteomic analysis revealed that the number of differentially expressed proteins (DEPs) was 285, of which 75 were upregulated and 210 were downregulated, and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways primarily comprised glycolysis, tricarboxylic acid cycle, fatty acid metabolism, secondary metabolite generation, and other pathways. Among the DEPs involved in PG inhibition of P. sojae are two closely related uncharacterized proteins encoded by PHYSODRAFT_522340 and PHYSODRAFT_344464, denoted PsFACL and PsCPT herein. The CRISPR/Cas9 knockout technique revealed that PsFACL and PsCPT were involved in the growth rate and pathogenicity. In addition, the results of gas chromatography-mass spectrometry (GC-MS) showed that there were differences in fatty acid levels between wild-type (WT) and CRISPR/Cas9 knockout transformants. Knocking out PsFACL and PsCPT resulted in the restriction of the synthesis and ß-oxidation of long-chain fatty acids, respectively. These suggest that PsFACL and PsCPT were also involved in the regulation of the fatty acid metabolism. Our results aid in understanding the mechanism underlying the inhibition of P. sojae growth by PG.

Correction to: Development of a novel strategy for fungal transformation based on a mutant locus conferring carboxin-resistance in Magnaporthe oryzae.

Following publication of the original article (Guo et al. 2016), the authors of Guo et al. (2016) would like to make a correction for a figure in their published article. An image in Fig. 4b (Lane 4, M3) were mistakenly used for strain M3. We checked the original images and replaced it with the correct one as showed below. We confirm that this change do not alter the findings of this work.

Pleiotropic roles of O-mannosyltransferase MoPmt4 in development and pathogenicity of Magnaporthe oryzae.

In fungi, O-mannosylation is one type of conserved protein modifications that add the carbohydrate residues to specific residues of target proteins by protein O-mannosyltransferases. Previously, three members of O-mannosyltransferases were identified in Magnaporthe oryzae, with MoPmt2 playing important roles in fungal growth and pathogenicity. However, the biological roles of the rest Pmt proteins remain unclear. In this study, to understand if O-mannosyltransferases are crucial for fungal pathogenicity of M. oryzae, the Pmt-coding genes MoPmt1 and MoPmt4 were separately disrupted and their roles in pathogenesis were analyzed. Of the two genes, only MoPmt4 is specifically required for full virulence of M. oryzae. Deletion of MoPmt4 resulted in defects on radial growth, with more branching hyphae and septa as compared to Guy11. The MoPmt4 mutant was severely impaired not only in conidiation, but also in both penetration and biotrophic invasion in susceptible rice plants. This mutant also had defects in suppression of host-derived ROS-mediated plant defense responses that might be ascribed from the reduced activities of extracellular enzymes. Furthermore, like their fungi counterparts, MoPmt4 localized in the ER and had O-mannosyltransferase activity. Domain disruption analysis indicated that mannosyltransferase activity regulated by PMT domain of MoPmt4 is crucial for fungal development and pathogenicity of M. oryzae. Taken together, these data suggest that MoPmt4 is a protein O-mannosyltransferase essential for fungal development and full virulence of M. oryzae.

Characterization of PGRP-S1 from the oriental armyworm, Mythimna separata.

Peptidoglycan is the key component forming the backbone of bacterial cell wall. It can be recognized by a group of pattern recognition receptors, known as peptidoglycan recognition proteins (PGRPs) in insects and higher animals. PGRPs may serve as immune receptors or N-acetylmuramoyl-L-alanine amidases (EC 3.5.1.28). Here, we report the characterization of a short PGRP, PGRP-S1, from the oriental armyworm, Mythimna separata. MsePGRP-S1 cDNA encodes a protein of 197 amino acids (aa) with a PGRP domain of about 150 aa. MsePGRP-S1 was expressed in several tissues of naïve larvae, including hemocytes, midgut, fat body and epidermis. Bacterial challenges caused variable changes in different tissues at the mRNA level. The recombinant protein bound strongly to Staphylococcus aureus and purified peptidoglycans from Staphylococcus aureus and Bacillus subtilis. It can inhibit the growth of gram-negative and gram-positive bacteria by disrupting bacterial surface. It can degrade peptidoglycans from Escherichia coli and Staphylococcus aureus. Taken together, these data demonstrate that M. separata PGRP-S1 is involved in defending against bacteria.

Correction to: Pleiotropic roles of O-mannosyltransferase MoPmt4 in development and pathogenicity of Magnaporthe oryzae.

In the original publication, Fig. 1 panel (b), the photo of MoPmt2-6 was incorrect. The correct Fig. 1 is shown below.

Correction to: MoGrr1, a novel F-box protein, is involved in conidiogenesis and cell wall integrity and is critical for the full virulence of Magnaporthe oryzae.

There is an error in the Original Publication. Two images were mistakenly edited in Fig.6 (panel (a)) and Fig.7 (panel (a). Please find below the corrected figures.

Antimicrobial activities of a proline-rich proprotein from Spodoptera litura.

Antimicrobial peptides (AMPs) are produced by the stimulated humoral immune system. Most mature AMPs contain less than 50 amino acid residues. Some of them are generated from proproteins upon microbial challenges. Here, we report the antimicrobial activities of a proline-rich proprotein, named SlLebocin1 (SlLeb1), from the tobacco cutworm Spodoptera litura. SlLebocin1 cDNA contains a 477-bp open reading frame (ORF). It is mainly expressed in hemocytes and the midgut in naïve larvae. The transcript level was significantly induced in hemocytes but repressed in the midgut and fat body by bacterial challenges. The proprotein contains 158 amino acids with 3 RXXR motifs that are characteristic of some Lepidopteral lebocin proproteins. Four peptides corresponding to the predicted processed fragments were synthesized chemically, and their antimicrobial activities against two Gram-negative and two Gram-positive bacterial strains were analyzed. The peptides showed differential antimicrobial activities. For Escherichia coli and Bacillus subtilis, only the C-terminal fragment (124-158) showed strong inhibitory effects. For Staphylococcus aureus, all peptides showed partial inhibitions. None of them inhibited Serratia marcescens. Bacterial morphologies were examined by the scanning electron microscopy and confocal laser scanning microscopy. The antimicrobial peptides either disrupted cellular membrane or inhibited cell division and caused elongated/enlarged morphologies. The results may provide ideas for designing novel antibiotics.

SsSm1, a Cerato-platanin family protein, is involved in the hyphal development and pathogenic process of Sclerotinia sclerotiorum.

The filamentous fungus Sclerotinia sclerotiorum is an important plant pathogen with a worldwide distribution. It can infect a wide variety of plants, causing serious disease in many types of crops, such as rapeseed, sunflower and soybean. Sclerotinia stem rot caused by this fungus affects main crops and has led to great economic loss. Elicitors are a group of compounds that inspire the host plant to produce an immune response against invading pathogens. This study describes a protein that has high homology with the Trichoderma elicitor Sm1 and was found in the genome of S. sclerotiorum. We named this protein SsSm1. To determine whether this protein has an elicitor function like its homology protein, we constructed a heterologous expression vector for SsSm1 and expressed it in Escherichia coli. The protein of heterologous expression led to the formation of lesions in tobacco that closely resemble hypersensitive response lesions. Transient expression of the encoding gene of SsSm1 in tobacco leaves also caused hypersensitive response. Then, RNA silencing was used to identify the function of SsSm1. The hyphal growth and pathogenicity of silenced transformants were shown to be obviously lagging and branched abnormally. Transformants produced less infection cushions and deformed sclerotiorum. In addition, SsSm1 silencing caused weak tolerance to NaCl, sorbitol and SDS, and the sensitivity of mutants to carbendazim was also significantly decreased. Based on the above results, we speculate that this protein may be related to the development of hyphae, infection cushions and sclerotiorum, but the specific molecular mechanism needs to be studied further.

Immune functions of insect ßGRPs and their potential application.

Insects rely completely on the innate immune system to sense the foreign bodies and to mount the immune responses. Germ-line encoded pattern recognition receptors play crucial roles in recognizing pathogen-associated molecular patterns. Among them, ß-1,3-glucan recognition proteins (ßGRPs) and gram-negative bacteria-binding proteins (GNBPs) belong to the same pattern recognition receptor family, which can recognize ß-1,3-glucans. Typical insect ßGRPs are comprised of a tandem carbohydrate-binding module in the N-terminal and a glucanase-like domain in the C-terminal. The former can recognize triple-helical ß-1,3-glucans, whereas the latter, which normally lacks the enzymatic activity, can recruit adapter proteins to initiate the protease cascade. According to studies, insect ßGRPs possess at least three types of functions. Firstly, some ßGRPs cooperate with peptidoglycan recognition proteins to recognize the lysine-type peptidoglycans upstream of the Toll pathway. Secondly, some directly recognize fungal ß-1,3-glucans to activate the Toll pathway and melanization. Thirdly, some form the 'attack complexes' with other immune effectors to promote the antifungal defenses. The current review will focus on the discovery of insect ßGRPs, functions of some well-characterized members, structure-function studies and their potential application.

Cloning and functional analysis of succinate dehydrogenase gene PsSDHA in Phytophthora sojae.

Succinate dehydrogenase (SDH) is one of the key enzymes of the tricarboxylic acid cycle (TCA cycle) and a proven target of fungicides for true fungi. To explore the roles of the SDHA gene in Phytophthora sojae, we first cloned PsSDHA to construct the PsSDHA silenced expression vector pHAM34-PsSDHA, and then utilized PEG to mediate the P. sojae protoplast transformation experiment. Through transformation screening, we obtained the silenced mutants A1 and A3, which have significant suppressive effect. Further study showed that the hyphae of the silenced mutant strains were shorter and more bifurcated; the growth of the silenced mutants was clearly inhibited in 10% V8 agar medium containing sodium chloride (NaCl), hydrogen peroxide (H2O2) or Congo Red, respectively. The pathogenicity of the silenced mutants was significantly reduced compared with the wild-type strain and the mock. The results could help us better to understand the position and function of SDH in P. sojae and provide a proven target of fungicides for the oomycete.

Development of a novel strategy for fungal transformation based on a mutant locus conferring carboxin-resistance in Magnaporthe oryzae.

The accurate manipulation of genomic integration of mutant alleles or fluorescent fusion-protein constructs is necessary for understanding of pathogenic mechanism of Magnaporthe oryzae. Recently, this can be achieved by integrating of exogenous DNA randomly into genome of this pathogen, but ectopic integration may result in alteration of gene expression or gene disruption due to unpredictable position effects and/or disruption of protein-coding regions. In this study, we establish a novel strategy for locus-specific integration of exogenous DNA via carboxin-resistance reconstitution by a point-mutation (H245L) on succinate dehydrogenase subunit Mosdi1. Independent transformants derived from the same reconstitution construct showed consistent fluorescent signal and undiversified phenotypes, including hyphae growth, conidiation and pathogenicity, in M. oryzae. Meanwhile, 96 % of all transformants integrate correctly into the Mosdi1 locus as a single copy. Furthermore, we provide a vector carrying yeast recombination cassette and thus allow assembly of multiple overlapping DNA fragments by yeast in vivo recombination for gene complementation and protein localization assay.