Use of Random T-DNA Mutagenesis in Identification of Gene UvPRO1, A Regulator of Conidiation, Stress Response, and Virulence in Ustilaginoidea virens

Lv, Bo; Zheng, Lu; Líu, Hao; Tang, Jintian; Hsiang, Tom; Huang, Jinbin

doi:10.3389/fmicb.2016.02086

Cited by 49 publications

(42 citation statements)

References 40 publications

(41 reference statements)

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“…Unlike many other plant pathogenic fungi, there are only limited molecular genetic studies in U. virens although its genome was sequenced and published in Zhang et al (2014) . To date, only the UvHOG1, UvSUN2, Uvt3277 , and UvPRO1 genes have been functionally characterized by deletion or disruption in this important plant pathogenic fungus ( Yu et al, 2015 ; Lv et al, 2016 ; Zheng et al, 2016 , 2017 ). One major bottleneck for molecular genetic studies with U. virens is its low homologous recombination frequency using the conventional gene replacement approaches.…”

Section: Introductionmentioning

confidence: 99%

“…One major bottleneck for molecular genetic studies with U. virens is its low homologous recombination frequency using the conventional gene replacement approaches. Mutants disrupted of the UvSUN2, Uvt3277 , and UvPRO1 genes were generated by random insertional mutagenesis instead of targeted gene deletion or disruption ( Yu et al, 2015 ; Lv et al, 2016 ; Zheng et al, 2017 ). For UvHOG1 , the only U. virens gene with mutants generated by targeted gene deletion, less than 0.5% of hygromycin-resistant transformants generated by Agrobacterium tumefaciens -mediated transformation (ATMT) were confirmed to true deletion mutants ( Zheng et al, 2016 ).…”

Section: Introductionmentioning

confidence: 99%

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Targeted Deletion of the USTA and UvSLT2 Genes Efficiently in Ustilaginoidea virens With the CRISPR-Cas9 System

Liang

Wang

et al. 2018

Front. Plant Sci.

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Ustilaginoidea virens is the causal agent of rice false smut, one of the major fungal diseases of rice. However, there are only limited molecular studies with this important pathogen due to the lack of efficient approaches for generating targeted gene disruption mutants. In this study, we used the CRISPR-Cas9 system to efficiently generate mutants deleted of the USTA ustiloxin and UvSLT2 MAP kinase genes. Three gRNA spacers of USTA, UA01, UA13, and UA21, were expressed with the RNAP III promoter of Gln-tRNA. For all of them, the homologous gene replacement frequency was higher when the Cas9 and gRNA constructs were transformed into U. virens on the same vector than sequentially. UA01, the spacer with the highest on-target score, had the highest knockout frequency of 90%, which was over 200 times higher than that of Agrobacterium tumefaciens-mediated transformation (ATMT) for generating ustA mutants. None of these USTA spacers had predicted off-targets with 1 or 2-nt variations. For predicted off-targets with 3 or 4-nt variations, mutations were not detected in 10 ustA mutants generated with spacer UA13 or UA21, indicating a relatively low frequency of off-target mutations in U. virens. For UvSLT2, the homologous gene replacement frequency was 50% with CRISPR-Cas9, which also was significantly higher than that of ATMT. Whereas ustA mutants had no detectable phenotypes, Uvslt2 mutants were slightly reduced in growth rate and reduced over 70% in conidiation. Deletion of UvSLT2 also increased sensitivity to cell wall stresses but tolerance to hyperosmotic or oxidative stresses. Taken together, our results showed that the CRISPR-Cas9 system can be used as an efficient gene replacement or editing approach in U. virens and the UvSlt2 MAP kinase pathway has a conserved role in cell wall integrity.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Targeted Deletion of the USTA and UvSLT2 Genes Efficiently in Ustilaginoidea virens With the CRISPR-Cas9 System

Liang

Wang

et al. 2018

Front. Plant Sci.

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“…Thus far, only few pathogenesis-related proteins have been identified and characterized in U. virens, including the hypothetical protein UvPro1, the low-affinity iron transporter Uvt3277, two protein kinases UvPmk1 and UvCDC2, the cyclic adenosine monophosphate signaling proteins adenylate cyclase UvAc1 and phosphodiesterase UvPdeH, the Bax inhibitor UvBI-1, the effector Scre2 (Uv_1261), two transcriptional factors UvHox2 and UvCom1, the phosphatase UvPsr1, and the autophagy-related protein UvAtg8 (Lv et al, 2016, Zheng et al, 2017, Fang et al, 2019, Guo et al, 2019, Tang et al, 2019, Chen et al, 2020. Among these, UvPmk1, UvCdc2, UvPro1, UvBI-1, UvAc1, UvPdeh, UvPsr1, UvCom1, UvHox2, and UvAtg8 are involved in both various stresses responses and pathogenesis of U. virens.…”

Section: Introductionmentioning

confidence: 99%

UvWHI2 is Required for Stress Response and Pathogenicity in Ustilaginonidea Virens

Meng¹,

Qiu²,

Xiong³

et al. 2020

Preprint

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Background: Ustilaginoidea virens causes rice false smut disease, which emerges as a worldwide disease of rice. At present, some stress response related genes have been identified in U. virens , but it is not clear whether and how defects of stress responses affect the pathogenesis processes of U. virens . To answer this question, the function of a general stress response factor Whi2 was analyzed in U. virens . Results: In this study, we identified UvWhi2 as a homolog of Saccharomyces cerevisiae Whi2 in U. virens. The relative expression level of UvWhi2 was significantly up-regulated during infection, suggesting that UvWhi2 may be involved in pathogenesis. Furthermore, knockout of UvWhi2 showed decreased the mycelial growth, increased in conidiation in the PS (potato sucrose) medium and a defect in pathogenicity. In addition, the RNA-Seq and phenotypic analysis showed that UvWHI2 is involved in response to oxidative, hyperosmotic, cell wall stresses, and nutrient limitation. Further studies revealed that the defects of stress responses of the ∆ Uvwhi2 mutant affected the formation of secondary spores on the nutrient limited surface and the rice surface, resulting in a significant reduction of pathogenicity of U. virens . Conclusions: Our results suggest that UvWhi2 is necessary for fungal growth, stress responses, and the formation of secondary spores in U. virens . In addition, the defects of stress responses could affect the formation of secondary spores on the rice surface, and then compromise the pathogenicity of U. virens .

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“…In addition, a few genes have been found to be associated with infection of U. virens in rice. For example, UvPRO1 encodes a C6 transcription factor, and disruption of the gene resulted in reduced growth rate, inability to sporulate, increased sensitivity to abiotic stresses, and abolishment of virulence (Lv et al 2016). UvSUN2 encodes a SUN domain-containing protein, and mutation in UvSUN2 led to altered cell wall construction, abnormal fungal growth and stress responses, as well as inability to infect rice (Yu et al 2015).…”

Section: Introductionmentioning

confidence: 99%

A core effector UV_1261 promotes Ustilaginoidea virens infection via spatiotemporally suppressing plant defense

Fan

et al. 2019

Phytopathol Res

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False smut is a destructive grain disease of rice worldwide, characterized by false smut balls formed in rice flowers. Here we identified a small secreted protein UV_1261 contributing to virulence of Ustilaginoidea virens, the causal agent of this disease. The sequence of UV_1261 was highly conserved among isolates of U. virens and absent in other fungi. UV_1261 encodes a protein targeted to plant chloroplasts. Its expression exhibited a bimodal pattern during pathogenesis. Ectopic expression of UV_1261 in Nicotiana benthamiana and Arabidopsis led to suppression of flg22-induced ROS burst, callose deposition, and expression of defense-related genes, as well as enhanced susceptibility to powdery mildew in Arabidopsis. Down-regulation of UV_1261 via exogenous siRNA treatment resulted in reduced number of false smut balls. Consistently, stably knocking-down UV_1261 caused less number of false smut balls associated with higher expression of defense-related genes in rice flower. Taken together, our data demonstrate that UV_1261 is a core effector of U. virens essential for virulence and suppressing defense in rice flower, and thus may serve as a potential molecular target for controlling rice false smut disease.

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Use of Random T-DNA Mutagenesis in Identification of Gene UvPRO1, A Regulator of Conidiation, Stress Response, and Virulence in Ustilaginoidea virens

Cited by 49 publications

References 40 publications

Targeted Deletion of the USTA and UvSLT2 Genes Efficiently in Ustilaginoidea virens With the CRISPR-Cas9 System

Targeted Deletion of the USTA and UvSLT2 Genes Efficiently in Ustilaginoidea virens With the CRISPR-Cas9 System

UvWHI2 is Required for Stress Response and Pathogenicity in Ustilaginonidea Virens

A core effector UV_1261 promotes Ustilaginoidea virens infection via spatiotemporally suppressing plant defense

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