Pinggen Xi

A Novel Multidomain Polyketide Synthase Is Essential for Zeamine Production and the Virulence of Dickeya zeae

Dickeya zeae is the causal agent of the rice foot rot disease, but its mechanism of infection remains largely unknown. In this study, we identified and characterized a novel gene designated as zmsA. The gene encodes a large protein of 2,346 amino acids in length, which consists of multidomains arranged in the order of N-terminus, β-ketoacyl synthase, acyl transferase, acyl carrier protein, β-ketoacyl reductase, dehydratase. This multidomain structure and sequence alignment analysis suggest that ZmsA is a member of the polyketide synthase family. Mutation of zmsA abolished antimicrobial activity and attenuated the virulence of D. zeae. To determine the relationship between antimicrobial activity and virulence, active compounds were purified from D. zeae EC1 and were structurally characterized. This led to identification of two polyamino compounds, i.e., zeamine and zeamine II, that were phytotoxins and potent antibiotics. These results have established the essential role of ZmsA in zeamine biosynthesis and presented a new insight on the molecular mechanisms of D. zeae pathogenicity.

Mitogen-Activated Protein Kinases Are Associated with the Regulation of Physiological Traits and Virulence in Fusarium oxysporum f. sp. cubense

Fusarium oxysporum f. sp. cubense (FOC) is an important soil-borne fungal pathogen causing devastating vascular wilt disease of banana plants and has become a great concern threatening banana production worldwide. However, little information is known about the molecular mechanisms that govern the expression of virulence determinants of this important fungal pathogen. In this study, we showed that null mutation of three mitogen-activated protein (MAP) kinase genes, designated as FoSlt2, FoMkk2 and FoBck1, respectively, led to substantial attenuation in fungal virulence on banana plants. Transcriptional analysis revealed that the MAP kinase signaling pathway plays a key role in regulation of the genes encoding production of chitin, peroxidase, beauvericin and fusaric acid. Biochemical analysis further confirmed the essential role of MAP kinases in modulating the production of fusaric acid, which was a crucial phytotoxin in accelerating development of Fusarium wilt symptoms in banana plants. Additionally, we found that the MAP kinase FoSlt2 was required for siderophore biosynthesis under iron-depletion conditions. Moreover, disruption of the MAP kinase genes resulted in abnormal hypha and increased sensitivity to Congo Red, Calcofluor White and H2O2. Taken together, these results depict the critical roles of MAP kinases in regulation of FOC physiology and virulence.

The mating-type locus b of the sugarcane smut Sporisorium scitamineum is essential for mating, filamentous growth and pathogenicity.

Sporisorium scitamineum is the causal agent of sugarcane smut, which is one of the most serious constraints to global sugarcane production. S. scitamineum and Ustilago maydis are two closely related smut fungi, that are predicted to harbor similar sexual mating processes/system. To elucidate the molecular basis of sexual mating in S. scitamineum, we identified and deleted the ortholog of mating-specific U. maydis locus b, in S. scitamineum. The resultant b-deletion mutant was defective in mating and pathogenicity in S. scitamineum. Furthermore, a functional b locus heterodimer could trigger filamentous growth without mating in S. scitamineum, and functionally replace the b locus in U. maydis in terms of triggering aerial filament production and forming solopathogenic strains, which do not require sexual mating prior to pathogenicity on the host plants.

Functional characterization of the gene FoOCH1 encoding a putative α-1,6-mannosyltransferase in Fusarium oxysporum f. sp. cubense.

Fusarium oxysporum f. sp. cubense (FOC) is the causal agent of banana Fusarium wilt and has become one of the most destructive pathogens threatening the banana production worldwide. However, few genes related to morphogenesis and pathogenicity of this fungal pathogen have been functionally characterized. In this study, we identified and characterized the disrupted gene in a T-DNA insertional mutant (L953) of FOC with significantly reduced virulence on banana plants. The gene disrupted by T-DNA insertion in L953 harbors an open reading frame, which encodes a protein with homology to α-1,6-mannosyltransferase (OCH1) in fungi. The deletion mutants (ΔFoOCH1) of the OCH1 orthologue (FoOCH1) in FOC were impaired in fungal growth, exhibited brighter staining with fluorescein isothiocyanate (FITC)-Concanavalin A, had less cell wall proteins and secreted more proteins into liquid media than the wild type. Furthermore, the mutation or deletion of FoOCH1 led to loss of ability to penetrate cellophane membrane and decline in hyphal attachment and colonization as well as virulence to the banana host. The mutant phenotypes were fully restored by complementation with the wild type FoOCH1 gene. Our data provide a first evidence for the critical role of FoOCH1 in maintenance of cell wall integrity and virulence of F. oxysporum f. sp. cubense.

Development of a sensitive nested-polymerase chain reaction (PCR) assay for the detection of Ustilago scitaminea

A species-specific polymerase chain reaction (PCR) assay was developed for rapid and accurate detection of Ustilago scitaminea, the causal agent of sugarcane smut disease. Based on nucleotide differences in the internal transcribed spacer (ITS) sequences of U. scitaminea, a pair of speciesspecific primers, SL1 (5`-CAGTGCACGAAAGTACCTGTGG-3`) and SR2 (5`CTAGGGCGGTGTTCAGAAGCAC-3`) was designed by using a panel of fungal and bacterial species as controls. The primers SL1/SR2 specifically amplified a unique PCR product about 530 bp in length from U. scitaminea strains with a detecting sensitivity at 200 fg of the fungal genomic DNA in a 25 μl reaction solution. To increase sensitivity, a nested-PCR protocol was further established, which used ITS4/ITS5 as the first-round primers followed by the primer pair SL1/SR2. This protocol increased the detection sensitivity by 10,000-fold compared to the PCR method and could detect the fungal DNA as low as 20 ag. The nested-PCR detected U. scitaminea from young sugarcane leaves with no visible smut disease symptoms. The findings from this study provide a sensitive and reliable technique for the early detection of U. scitaminea, which would be useful for sugarcane quarantine and production of germfree seedcanes.

A Puf RNA-binding protein encoding gene PlM90 regulates the sexual and asexual life stages of the litchi downy blight pathogen Peronophythora litchii

Sexual and asexual reproduction are two key processes in the pathogenic cycle of many filamentous pathogens. However in Peronophythora litchii, the causal pathogen for the litchi downy blight disease, critical regulator(s) of sexual or asexual differentiation has not been elucidated. In this study, we cloned a gene named PlM90 from P. litchii, which encodes a putative Puf RNA-binding protein. We found that PlM90 was highly expressed during asexual development, and much higher than that during sexual development, while relatively lower during cyst germination and plant infection. By polyethylene glycol (PEG)-mediated protoplast transformation, we generated three PlM90-silenced transformants and found a severely impaired ability in sexual spore production and a delay in stages of zoospore release and encystment. However, the pathogenicity of P. litchii was not affected by PlM90-silencing. Therefore we conclude that PlM90 specifically regulates the sexual and asexual differentiation of P. litchii.

Identification and application of a unique genetic locus in diagnosis of Fusarium oxysporum f. sp. cubense tropical race 4

Abstract Fusarium oxysporum f. sp. cubense, particularly the tropical race 4 (TR4), is the most destructive fungal pathogen of banana and can cause significant economic losses. However, the molecular mechanisms underlying the pathogenicity and virulence of the fungus are poorly understood. In this study, we characterized the mutant W2987, which was derived through T-DNA insertional mutagenesis of a wild-type TR4 isolate. W2987 showed slower growth, produced fewer conidia and was less virulent than the wild-type strain. Thermal asymmetric interlaced-PCR (TAIL-PCR) analysis showed that the T-DNA was inserted in the 3′ untranslated region (UTR) of an open reading frame (ORF) in W2987. BLAST search results indicated that the ORF encodes a hypothetical protein with unknown function. Phylogenetic analysis of the gene sequences from various Fusarium isolates showed that the F. oxysporum f. sp. cubense TR4 isolates were distinct from other F. oxysporum isolates. One pair of primers based on the 3′ UTR sequence of the putative virulence gene amplified a specific PCR product from the F. oxysporum f. sp. cubense TR4 isolates but not from other races and Fusarium species. This result indicates that this genetic locus possibly associated with virulence is unique and reliable for diagnosis and detection of F. oxysporum f. sp. cubense TR4.

A Dual-Color Imaging System for Sugarcane Smut Fungus Sporisorium scitamineum

The life cycle of the sugarcane smut fungus Sporisorium scitamineum is a multistep process. Haploid sporidia of compatible (MAT-1 versus MAT-2) mating types fuse to generate pathogenic dikaryotic hyphae to infect the host. Within the host tissues, diploid teliospores are formed and induce a characteristic sorus that looks like a black whip. The diploid teliospores germinate to form haploid sporidia by meiosis. In order to monitor fungal development throughout the whole life cycle, we expressed the green fluorescent protein (GFP) and red fluorescent protein (RFP) in S. scitamineum MAT-1 and MAT-2 sporidia, respectively. Observation by epifluorescence microscope showed that conjugation tube formation and sporidia fusion occurred at 4 to 8 h, and formation of dikaryotic filaments was detected at 12 h after mating. The resultant teliospores, with diffused GFP and RFP, underwent meiosis as demonstrated by septated hypha with single fluorescent signal. We demonstrated that GFP- and RFP-tagged strains can be used to study the life cycle development of the fungal pathogen S. scitamineum, including the sexual mating and meiosis events. This dual-color imaging system would be a valuable tool for investigation of biotic and abiotic factors that might affect the fungal life cycle development and pathogenesis.

A Destructive Leaf Spot and Blight Caused by Alternaria kareliniae sp. nov. on a Sand-Stabilizing Plant, Caspian Sea Karelinia

Leaf spots and stem lesions causing widespread mortality of Caspian Sea karelinia (Karelinia caspia) were observed in desert regions of Xinjiang Uyghur Autonomous Region, China. Fifteen samples were collected from five widely distributed counties of Tarim and Junggar Basins in 2016. The pathogen was identified using morphological observations and phylogenetic analyses based on combined partial sequences from seven genes (Alt a 1, ATPase, calmodulin, glyceraldehyde 3-phosphate dehydrogenase, internal transcribed spacer, RNA polymerase II, and translation elongation factor 1), and placed as a new species: Alternaria kareliniae sp. nov. in section Dianthicola. The fungus has a small conidium (24.3 to) 29.1 to 64.8 (to 75.8) by (9.3 to) 12.4 to 16.5 (to 21.7) μm with a long beak (130 to) 183.9 to 350.4 (to 378.2) μm, as well as four to eight transverse septa, which differs significantly from other species of Alternaria section Dianthicola. On potato carrot agar, it grew significantly more slowly than others of this section. Pathogenicity tests showed that the fungus could infect leaves and stems of K. caspia and cause the same symptoms as those observed in the field. The fungus was reisolated from inoculated leaves and stems of the host. The disease in desert regions appears to be increasing, and it may have future negative implications for desert ecology in these areas. Future research should concentrate on elucidating the disease cycle and disease management alternatives.

Antifungal Activity of Natural Volatile Organic Compounds against Litchi Downy Blight Pathogen Peronophythora litchii

Litchi (Litchi chinensis Sonn.) is a commercially important fruit but its production and quality are restricted by litchi downy blight, caused by the oomycete pathogen Peronophythora litchii Chen. Volatile substances produced by a biocontrol antinomycetes Streptomyces fimicarius BWL-H1 could inhibited P. litchii growth and development both in vitro and in detached litchi leaf and fruit infection assay. Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM) analyses indicated that volatile organic compounds (VOCs) from BWL-H1 resulted in severe damage to the endomembrane system and cell wall of P. litchii cells in vitro and abnormal morphology of appressoria, as well as deformed new hyphae in infection process. VOCs could suppress mycelial growth, sporulation, while with no obvious effect on sporangia germination. Based on gas chromatography-mass spectrophotometric analyses, 32 VOCs were identified from S. fimicarius BWL-H1, the most abundant of which was phenylethyl alcohol. Eight VOCs, including phenylethyl alcohol, ethyl phenylacetate, methyl anthranilate, α-copaene, caryophyllene, humulene, methyl salicylate and 4-ethylphenol, that are commercially available, were purchased and their bioactivity was tested individually. Except for humulene, the other seven tested volatile compounds shown strong inhibitory activity against mycelial growth, sporulation, sporangia germination and germ-tube growth of P. litchii. Especially, 4-ethylphenol showed the highest inhibitory effect on sporulation at a very low concentration of 2 µL/L. Overall, our results provided a better understanding of the mode of action of volatiles from BWL-H1 on P. litchii, and showed that volatiles from BWL-H1 have the potential for control of postharvest litchi downy blight.