Survival of Pseudomonas syringae pv. actinidiae in detached kiwifruit leaves at different environmental conditions.

Pseudomonas syringae pv. actinidiae (Psa) is the causal agent of kiwifruit canker, a serious threat to commercial kiwifruit production worldwide. Studies of the movement path and the survival time of Psa in the host are crucial for integrated management programs. Hence, we used Psa with GFPuv gene (Psa-GFPuv) strain to investigate the movement path of Psa in leaves and branches, and the survival time of Psa in leaves under different environmental conditions. We found that the pathogen Psa spread longitudinally in the branches and leaves rather than transverse path. Additionally, the survival time of bacteria in fallen leaves under different environmental conditions were simulated by the way of Psa infecting the detached kiwifruit leaves. Psa survives the longest, up to 43 days in detached kiwifruit leaves with high humidity (above 80%) at 5 °C, and up to 32 days with low humidity (20%). At 15 °C, the Psa can survive in detached kiwifruit leaves for 20-30 days with increasing humidity. At 25 °C, it can only survive for 3 days with low humidity (20%) and 15 days with high humidity (above 80%). Furthermore, the population growth experiments showed that bacterial growth of Psa was more favorable in detached kiwifruit leaves with above 80% humidity at 5 °C. These results suggest that the survival condition of Psa in detached kiwifruit leaves is significantly affected by environmental conditions, and provide the basis for the control timing and technology of kiwifruit canker.

Indole-3-Carboxylic Acid From the Endophytic Fungus Lasiodiplodia pseudotheobromae LPS-1 as a Synergist Enhancing the Antagonism of Jasmonic Acid Against Blumeria graminis on Wheat.

The discovery of natural bioactive compounds from endophytes or medicinal plants against plant diseases is an attractive option for reducing the use of chemical fungicides. In this study, three compounds, indole-3-carbaldehyde, indole-3-carboxylic acid (3-ICA), and jasmonic acid (JA), were isolated from the EtOAc extract of the culture filtrate of the endophytic fungus Lasiodiplodia pseudotheobromae LPS-1, which was previously isolated from the medicinal plant, Ilex cornuta. Some experiments were conducted to further determine the antifungal activity of these compounds on wheat powdery mildew. The results showed that JA was much more bioactive than indole-3-carbaldehyde and 3-ICA against Blumeria graminis, and the disease severity caused by B. graminis decreased significantly with the concentration increase of JA treatment. The assay of the interaction of 3-ICA and JA indicated that there was a significant synergistic effect between the two compounds on B. graminis in each of the ratios of 3-ICA to JA (3-ICA:JA) ranging from 1:9 to 9:1. When the compound ratio of 3-ICA to JA was 2:8, the synergistic coefficient was the highest as 22.95. Meanwhile, a histological investigation indicated that, under the treatment of JA at 500 µg/ml or 3-ICA:JA (2:8) at 40 µg/ml, the appressorium development and haustorium formation of B. graminis were significantly inhibited. Taken together, we concluded that JA plays an important role in the infection process of B. graminis and that 3-ICA as a synergist of JA enhances the antagonism against wheat powdery mildew.

The influence of rhizosphere soil fungal diversity and complex community structure on wheat root rot disease.

Wheat root rot disease due to soil-borne fungal pathogens leads to tremendous yield losses worth billions of dollars worldwide every year. It is very important to study the relationship between rhizosphere soil fungal diversity and wheat roots to understand the occurrence and development of wheat root rot disease. A significant difference in fungal diversity was observed in the rhizosphere soil of healthy and diseased wheat roots in the heading stage, but the trend was the opposite in the filling stage. The abundance of most genera with high richness decreased significantly from the heading to the filling stage in the diseased groups; the richness of approximately one-third of all genera remained unchanged, and only a few low-richness genera, such as Fusarium and Ceratobasidium, had a very significant increase from the heading to the filling stage. In the healthy groups, the abundance of most genera increased significantly from the heading to filling stage; the abundance of some genera did not change markedly, or the abundance of very few genera increased significantly. Physical and chemical soil indicators showed that low soil pH and density, increases in ammonium nitrogen, nitrate nitrogen and total nitrogen contributed to the occurrence of wheat root rot disease. Our results revealed that in the early stages of disease, highly diverse rhizosphere soil fungi and a complex community structure can easily cause wheat root rot disease. The existence of pathogenic fungi is a necessary condition for wheat root rot disease, but the richness of pathogenic fungi is not necessarily important. The increases in ammonium nitrogen, nitrate nitrogen and total nitrogen contributed to the occurrence of wheat root rot disease. Low soil pH and soil density are beneficial to the occurrence of wheat root rot disease.

Leucine biosynthesis is required for infection-related morphogenesis and pathogenicity in the rice blast fungus Magnaporthe oryzae.

The rice blast fungus Magnaporthe oryzae causes one of the most devastating crop diseases world-wide and new control strategies for blast disease are urgently required. We have used insertional mutagenesis in M. oryzae to define biological processes that are critical for blast disease. Here, we report the identification of LEU2A by T-DNA mutagenesis, which putatively encodes 3-isopropylmalate dehydrogenase (3-IPMDH) required for leucine biosynthesis, implicating that synthesis of this amino acid is required for fungal pathogenesis. M. oryzae contains a further predicted 3-IPMDH gene (LEU2B), two 2-isopropylmalate synthase (2-IPMS) genes (LEU4 and LEU9) and an isopropylmalate isomerase (IPMI) gene (LEU1). Targeted gene deletion mutants of LEU1, LEU2A or LEU4 are leucine auxotrophs, and severely defective in pathogenicity. All phenotypes associated with mutants lacking LEU1, LEU2A or LEU4 could be overcome by adding exogenous leucine. The expression levels of LEU1, LEU2A or LEU4 genes were significantly down-regulated by deletion of the transcription factor gene LEU3, an ortholog of Saccharomyces cerevisiae LEU3. We also functionally characterized leucine biosynthesis genes in the wheat pathogen Fusarium graminearum and found that FgLEU1, FgLEU3 and FgLEU4 are essential for wheat head blight disease, suggesting that leucine biosynthesis in filamentous fungal pathogens may be a conserved factor for fungal pathogenicity and, therefore, a potential target for disease control.

The putative deubiquitinating enzyme MoUbp4 is required for infection-related morphogenesis and pathogenicity in the rice blast fungus Magnaporthe oryzae.

Ubiquitination is a key regulatory mechanism that affects numerous important biological processes, including cellular differentiation and pathogenesis in eukaryotic cells. Attachment of proteins to ubiquitin is reversed by specialized proteases, deubiquitinating enzymes (DUBs), which are essential for precursor processing, maintaining ubiquitin homeostasis and promoting protein degradation by recycling ubiquitins. Here, we report the identification of a novel non-pathogenic T-DNA-tagged mutant T612 of Magnaporthe oryzae with a single insertion in the second exon of MoUBP4, which encodes a putative ubiquitin carboxyl-terminal hydrolase. Targeted gene deletion mutants of MoUBP4 are significantly reduced in mycelial growth, conidiation, and increased in tolerance to SDS and CR (Congo red) cell-wall damage. The ΔMoubp4 mutants are blocked in penetration and invasive growth, which results in the loss of pathogenicity. Many conidia produced by the ΔMoubp4 mutants are unable to form appressoria and mobilization and degradation of glycogen and lipid droplets are significantly delayed. Moreover, immunohybridization analysis revealed that total protein ubiquitination levels of the null mutants were significantly increased, indicating that MoUbp4 functions as a deubiquitination enzyme. Taken together, we conclude that MoUbp4 is required for deubiquitination, infection-related morphogenesis and pathogenicity in M. oryzae.

Genome-wide functional analysis reveals that autophagy is necessary for growth, sporulation, deoxynivalenol production and virulence in Fusarium graminearum.

Autophagy is a conserved cellular recycling and trafficking pathway in eukaryotic cells and has been reported to be important in the virulence of a number of microbial pathogens. Here, we report genome-wide identification and characterization of autophagy-related genes (ATGs) in the wheat pathogenic fungus Fusarium graminearum. We identified twenty-eight genes associated with the regulation and operation of autophagy in F. graminearum. Using targeted gene deletion, we generated a set of 28 isogenic mutants. Autophagy mutants were classified into two groups by differences in their growth patterns. Radial growth of 18 Group 1 ATG mutants was significantly reduced compared to the wild-type strain PH-1, while 10 Group 2 mutants grew normally. Loss of any of the ATG genes, except FgATG17, prevented the fungus from causing Fusarium head blight disease. Moreover, subsets of autophagy genes were necessary for asexual/sexual differentiation and deoxynivalenol (DON) production, respectively. FgATG1 and FgATG5 were investigated in detail and showed severe defects in autophagy. Taken together, we conclude that autophagy plays a critical role in growth, asexual/sexual sporulation, deoxynivalenol production and virulence in F. graminearum.

The cyclin dependent kinase subunit Cks1 is required for infection-associated development of the rice blast fungus Magnaporthe oryzae.

Cell cycle regulation is pivotal for proper cell division and cellular differentiation in eukaryotic cells. The central regulators that govern eukaryotic cell cycle progression are cyclin-dependent kinases (CDKs) and their partners. Here, we report that Magnaporthe oryzae CKS1 encodes a cyclin-dependent kinase subunit, which plays a significant role in regulation of plant infection. We demonstrate that CKS1 is a functional homolog of CKS1/SUC1 and can physically interact with the CDK protein Cdc28, and Som1, a downstream regulator of the cyclic AMP-dependent Protein Kinase A pathway. CKS1 deletion mutants are severely impaired in hyphal growth, sexual reproduction, melanin pigmentation and conidiogenesis. Cks1 mutants are able to form appressoria from hyphal tips, but these are unable to re-polarize, and rice infection is impaired. CKS1 also affects chitin and glucan synthase activity during cell wall differentiation and fungal hydrophobin function. CKS1, therefore, encodes a conserved CDK-binding partner, essential for appressorium-mediated plant infection by the rice blast fungus.

Identification and characterization of the peroxin 1 gene MoPEX1 required for infection-related morphogenesis and pathogenicity in Magnaporthe oryzae.

Peroxisomes are required for pathogenicity in many phytopathogenic fungi, but the relationships between fungal pathogenicity and peroxisomal function are not fully understood. Here, we report the identification of a T-DNA insertional mutant C445 of Magnaporthe oryzae, which is defective in pathogenicity. Analysis of the mutation confirmed an insertion into the gene MoPEX1, which encodes a putative homologue to peroxin 1. Targeted gene deletion mutants of MoPEX1 were nonpathogenic and were impaired in vegetative growth, conidiation, and appressorium formation. ΔMopex1 mutants formed abnormal, less pigmented, and nonfunctional appressoria, but they were unable to penetrate plant cuticle. The ΔMopex1 mutants were defective in the utilization of fatty acids (e.g., olive oil and Tween-20). Moreover, deletion of MoPEX1 significantly impaired the mobilization and degradation of lipid droplets during appressorium development. Interestingly, deletion of MoPEX1 blocked the import of peroxisomal matrix proteins. Analysis of an M. oryzae strain expressing GFP-MoPEX1 and RFP-PTS1 fusions revealed that MoPex1 localizes to peroxisomes. Yeast two hybrid experiments showed that MoPex1 physically interacts with MoPex6, a peroxisomal matrix protein important for fungal morphogenesis and pathogenicity. Taken together, we conclude that MoPEX1 plays important roles in peroxisomal function and is required for infection-related morphogenesis and pathogenicity in M. oryzae.

ZNF1 Encodes a Putative C2H2 Zinc-Finger Protein Essential for Appressorium Differentiation by the Rice Blast Fungus Magnaporthe oryzae.

The rice blast fungus Magnaporthe oryzae forms specialized infection structures called appressoria which are essential for gaining entry to plant tissue. Here, we report the identification of a novel nonpathogenic T-DNA-tagged mutant XF696 of M. oryzae with a single insertion in the promoter of ZNF1, which encodes a putative transcription factor (TF). Targeted gene deletion mutants of ZNF1 are nonpathogenic and unable to develop appressoria. However, Δznf1 mutants still respond to exogenous cyclic AMP on hydrophilic surfaces and can sense hydrophobic surfaces, initiating the differentiation of germ tubes. Interestingly, Δznf1 mutants also produce significantly more conidia compared with the isogenic wild-type strain. Quantitative reverse-transcription polymerase chain reaction analysis and green fluorescent protein fusion experiments revealed that expression of ZNF1 was highly induced during germination and appressorium development in M. oryzae and potentially regulated by the Pmk1 mitogen-activated protein kinase pathway. We observed that Δznf1 mutants are affected in mitosis and impaired in mobilization and degradation of lipid droplets and glycogen reserves during appressorium differentiation. Site-directed mutagenesis confirmed that three of the four C2H2 zinc-finger domains are essential for the function of Znf1. Taken together, we conclude that a C2H2 zinc-finger TF encoded by ZNF1 is essential for appressorium development by the rice blast fungus.

FgRIC8 is involved in regulating vegetative growth, conidiation, deoxynivalenol production and virulence in Fusarium graminearum.

Proteins of the resistance to inhibitors of cholinesterase 8 (Ric8) group act as guanine nucleotide exchange factors (GEFs) and play important roles in regulating G-protein signaling in animals. In filamentous fungi, putative Ric8 orthologs have so far been identified in Magnaporthe oryzae, Neurospora crassa, Aspergillus nidulans and Aspergillus fumigatus. Here, we report the functional investigation of a potential RIC8 ortholog (FgRIC8) in the wheat head blight pathogen Fusarium graminearum. Targeted gene deletion mutants of FgRIC8 exhibited a significant reduction in vegetative growth, conidiation, pigment production as well as deoxynivalenol (DON) biosynthesis. Pathogenicity assays using a point-inoculated spikelet approach showed that the mutants were severely impaired in virulence on flowering wheat heads. Quantitative RT-PCR analysis revealed that genes encoding F. graminearum Gα (FgGpa1 and FgGpa3), Gß (FgGpb1) and Gγ (FgGpg1) subunits were significantly down-regulated in Fgric8 mutants. Moreover, we showed that FgRic8 physically interacts with both FgGpa1 and FgGpa3, but not FgGpa2, in yeast two-hybrid assays. The intracellular cAMP levels in Fgric8 mutants were significantly decreased compared to the isogenic wild-type strain. Taken together, our results indicate that FgRic8 plays critical roles in fungal development, secondary metabolism and virulence in F. graminearum and may act as a regulator of G protein alpha subunits.

The putative Gγ subunit gene MGG1 is required for conidiation, appressorium formation, mating and pathogenicity in Magnaporthe oryzae.

Heterotrimeric G-proteins play key roles in the transduction of extracellular signals to various downstream effectors in eukaryotes. In our previous study, a T-DNA insertional mutant A1-412, in which the promoter of a putative Gγ subunit gene MGG1 was disrupted, was impaired in asexual/sexual sporulation, appressorium formation, and pathogenicity in Magnaporthe oryzae. Here the roles of MGG1 in regulating fungal development and plant infection were further investigated and verified using a gene deletion strategy. Targeted gene deletion mutants of MGG1 exhibited similar phenotypes to those of A1-412. The Δmgg1 mutants were unable to differentiate appressorium on hydrophobic surfaces and nonpathogenic to susceptible hosts. The defects of the Δmgg1 mutants in appressorium formation were partially restored by adding exogenous cAMP or IBMX (a phosphodiesterase inhibitor), although the induced appressoria were still nonfunctional. Expressing Mgg1-GFP fusion protein in an Δmgg1 mutant could complement all phenotypes of the mutant, and bright GFP fluorescence was observed at the periphery of fungal cells, indicating that Mgg1 mainly localizes to plasma membrane. Quantitative RT-PCR analysis revealed that deletion of MGG1 resulted in a significant reduction in mRNA levels of the genes encoding Gα (MagA, MagB, and MagC), Gß (Mgb1), and adenylate cyclase (Mac1). Moreover, intracellular cAMP accumulation was significantly reduced in Δmgg1 mutants compared to that in the wild-type strain. Taken together, our results suggested that Gγ subunit Mgg1 might act upstream of cAMP signaling pathway and play critical roles in regulation of conidiation, appressorium formation, mating, and plant infection in M. oryzae.

Characterisation of four LIM protein-encoding genes involved in infection-related development and pathogenicity by the rice blast fungus Magnaporthe oryzae.

LIM domain proteins contain contiguous double-zinc finger domains and play important roles in cytoskeletal re-organisation and organ development in multi-cellular eukaryotes. Here, we report the characterization of four genes encoding LIM proteins in the rice blast fungus Magnaporthe oryzae. Targeted gene replacement of either the paxillin-encoding gene, PAX1, or LRG1 resulted in a significant reduction in hyphal growth and loss of pathogenicity, while deletion of RGA1 caused defects in conidiogenesis and appressorium development. A fourth LIM domain gene, LDP1, was not required for infection-associated development by M. oryzae. Live cell imaging revealed that Lrg1-GFP and Rga1-GFP both localize to septal pores, while Pax1-GFP is present in the cytoplasm. To explore the function of individual LIM domains, we carried out systematic deletion of each LIM domain, which revealed the importance of the Lrg1-LIM2 and Lrg1-RhoGAP domains for Lrg1 function and overlapping functions of the three LIM domains of Pax1. Interestingly, deletion of either PAX1 or LRG1 led to decreased sensitivity to cell wall-perturbing agents, such as Congo Red and SDS (sodium dodecyl sulfate). qRT-PCR analysis demonstrated the importance of both Lrg1 and Pax1 to regulation of genes associated with cell wall biogenesis. When considered together, our results indicate that LIM domain proteins are key regulators of infection-associated morphogenesis by the rice blast fungus.

The MET13 methylenetetrahydrofolate reductase gene is essential for infection-related morphogenesis in the rice blast fungus Magnaporthe oryzae.

Methylenetetrahydrofolate reductases (MTHFRs) play a key role in the biosynthesis of methionine in both prokaryotic and eukaryotic organisms. In this study, we report the identification of a novel T-DNA-tagged mutant WH672 in the rice blast fungus Magnaporthe oryzae, which was defective in vegetative growth, conidiation and pathogenicity. Analysis of the mutation confirmed a single T-DNA insertion upstream of MET13, which encodes a 626-amino-acid protein encoding a MTHFR. Targeted gene deletion of MET13 resulted in mutants that were non-pathogenic and significantly impaired in aerial growth and melanin pigmentation. All phenotypes associated with Δmet13 mutants could be overcome by addition of exogenous methionine. The M. oryzae genome contains a second predicted MTHFR-encoding gene, MET12. The deduced amino acid sequences of Met13 and Met12 share 32% identity. Interestingly, Δmet12 mutants produced significantly less conidia compared with the isogenic wild-type strain and grew very poorly in the absence of methionine, but were fully pathogenic. Deletion of both genes resulted in Δmet13Δmet12 mutants that showed similar phenotypes to single Δmet13 mutants. Taken together, we conclude that the MTHFR gene, MET13, is essential for infection-related morphogenesis by the rice blast fungus M. oryzae.

Two novel transcriptional regulators are essential for infection-related morphogenesis and pathogenicity of the rice blast fungus Magnaporthe oryzae.

The cyclic AMP-dependent protein kinase A signaling pathway plays a major role in regulating plant infection by the rice blast fungus Magnaporthe oryzae. Here, we report the identification of two novel genes, MoSOM1 and MoCDTF1, which were discovered in an insertional mutagenesis screen for non-pathogenic mutants of M. oryzae. MoSOM1 or MoCDTF1 are both necessary for development of spores and appressoria by M. oryzae and play roles in cell wall differentiation, regulating melanin pigmentation and cell surface hydrophobicity during spore formation. MoSom1 strongly interacts with MoStu1 (Mstu1), an APSES transcription factor protein, and with MoCdtf1, while also interacting more weakly with the catalytic subunit of protein kinase A (CpkA) in yeast two hybrid assays. Furthermore, the expression levels of MoSOM1 and MoCDTF1 were significantly reduced in both Δmac1 and ΔcpkA mutants, consistent with regulation by the cAMP/PKA signaling pathway. MoSom1-GFP and MoCdtf1-GFP fusion proteins localized to the nucleus of fungal cells. Site-directed mutagenesis confirmed that nuclear localization signal sequences in MoSom1 and MoCdtf1 are essential for their sub-cellular localization and biological functions. Transcriptional profiling revealed major changes in gene expression associated with loss of MoSOM1 during infection-related development. We conclude that MoSom1 and MoCdtf1 functions downstream of the cAMP/PKA signaling pathway and are novel transcriptional regulators associated with cellular differentiation during plant infection by the rice blast fungus.

A sterol 14α-demethylase is required for conidiation, virulence and for mediating sensitivity to sterol demethylation inhibitors by the rice blast fungus Magnaporthe oryzae.

The Magnaporthe oryzae genome contains two homologous CYP51 genes, MoCYP51A and MoCYP51B, that putatively encode sterol 14α-demethylase enzymes. Targeted gene deletion mutants of MoCYP51A were morphologically indistinguishable from the isogenic wild type M. oryzae strain Guy11 in vegetative culture, but were impaired in both conidiation and virulence. Deletion of MoCYP51B did not result in any obvious phenotypic changes compared with Guy11. The Δmocyp51A mutants were also highly sensitive to sterol demethylation inhibitor (DMI) fungicides, while Δmocyp51B mutants were unchanged in their sensitivity to these fungicides. Expression of both MoCYP51A and MoCYP51B was significantly induced by exposure to DMI fungicides. Analysis of intracellular localization of MoCyp51A showed that MoCyp51A was mainly localized to the cytoplasm of hyphae and conidia. Taken together, our results indicate that MoCYP51A is required for efficient conidiogenesis, full virulence and for mediating DMI sensitivity by the rice blast fungus.