Agrobacterium tumefaciens -mediated transformation of the entomopathogenic fungus Nomuraea rileyi

Shao, Changwen; Yin, Yue; Qi, Zhaoran; Li, Ren; Song, Zhimin; Li, Yan; Wang, Zhongkang

doi:10.1016/j.fgb.2015.08.002

Cited by 21 publications

(13 citation statements)

References 36 publications

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“…In this study, using the transformation protocol recently developed in our laboratory38, we examined the functions of the Hog1- and Slt2-type MAPKs in M. rileyi in response to stress, and their roles in morphology and pathogenicity. Consistent with findings in other fungi, two MAPKs are important in developmental processes and in pathogenesis26283940.…”

Section: Discussionmentioning

confidence: 99%

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The high osmotic response and cell wall integrity pathways cooperate to regulate morphology, microsclerotia development, and virulence in Metarhizium rileyi

Song

Yin

Shen

et al. 2016

Sci Rep

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Microsclerotia (MS) formation was successfully induced in Metarhizium rileyi under changing liquid culture conditions. Mitogen-activated protein kinases (MAPKs) play important roles in fungal development and in coordinating many stress responses. To investigate how M. rileyi transduces growth stress and regulates MS differentiation, we characterized the roles of two MAPKs, Hog1- and Slt2-type orthologues, in M. rileyi. Compared with the wild-type strain, the deletion mutants of Mrhog1 (ΔMrhog1) and Mrslt2 (ΔMrslt2) delayed germination and vegetative growth, displayed sensitivities to various stress, and produced morphologically abnormal clones. The ΔMrhog1 and ΔMrslt2 mutants significantly reduced conidial (42–99%) and MS (96–99%) yields. A transcriptional analysis showed that the two MAPKs regulate MS development in a cooperative manner. Insect bioassays revealed that ΔMrhog1 and ΔMrslt2 had decreased virulence levels in topical (36–56%) and injection (78–93%) bioassays. Our results confirmed the roles of MrHog1 and MrSlt2 in sensing growth-related stress and in regulating MS differentiation.

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Section: Discussionmentioning

confidence: 99%

“…The pPZP-Hph-Mrhog1 and pPZP-Hph-Mrslt2 plasmids were proliferated in E. coli DH5α and transformed into the WT strain using Agrobacterium -mediated transformation38. Putative gene disruption mutants were screened on SMAY supplemented with 450 μg/ml Hyg B.…”

Section: Methodsmentioning

confidence: 99%

The high osmotic response and cell wall integrity pathways cooperate to regulate morphology, microsclerotia development, and virulence in Metarhizium rileyi

Song

Yin

Shen

et al. 2016

Sci Rep

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“…Escherichia coli DH5α (Invitrogen, Shanghai, China) was used for recombinant DNA manipulation and Agrobacterium tumefaciens AGL‐1 (Invitrogen, Shanghai, China) for fungal transformations. Both were cultured as previously described (Shao et al ., 2015; Song et al ., 2016b). …”

Section: Methodsmentioning

confidence: 99%

“…This study seeks to further elucidate the role of MrMsn2 in dimorphism transition, conidiation, virulence and MS formation by phenotypic analyses of deletion/rescue mutants constructed previously (Shao et al ., 2015; Song et al ., 2016b). We found that the absence of MrMsn2 resulted in increased yeast‐to‐hypha transition rate, enhanced conidiation capacity, aggravated pigmentation and induced or suppressed expression of target genes involved in the important phenotypes of M. rileyi , as presented below.…”

Section: Introductionmentioning

confidence: 99%

A transcription factor, MrMsn2, in the dimorphic fungus Metarhizium rileyi is essential for dimorphism transition, aggravated pigmentation, conidiation and microsclerotia formation

Song

Yang

Xin

et al. 2018

Microbial Biotechnology

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SummaryMicrosclerotia (MS) are pseudoparenchymatous aggregations of hyphae of fungi that can be induced in liquid culture for biocontrol applications. Previously, we determined that the high‐osmolarity glycerol (HOG) signalling pathway was involved in regulating MS development in the dimorphic insect pathogen Metarhizium rileyi. To further investigate the mechanisms by which the signalling pathway is regulated, we characterized the transcriptional factor MrMsn2, a homologue of the yeast C2H2 transcriptional factor Msn2, which is predicted to function downstream of the HOG pathway in M. rileyi. Compared with wild‐type and complemented strains, disruption of MrMsn2 increased the yeast‐to‐hypha transition rate, enhanced conidiation capacity and aggravated pigmentation in M. rileyi. The ▵MrMsn2 mutants were sensitive to stress, produced morphologically abnormal clones and had significantly reduced MS formation and decreased virulence levels. Digital expression profiling revealed that genes involved in antioxidation, pigment biosynthesis and ion transport and storage were regulated by MrMsn2 during conidia and MS development. Taken together, our findings confirm that MrMsn2 controlled the yeast‐to‐hypha transition, conidia and MS formation, and virulence.

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“…The resulting clones were introduced into Agrobacterium strain AGL1 for fungal transformation. Agrobacterium tumefaciens mediated transformation (ATMT) of N.rileyi CQNr01 with pPZP-Hph-NrSidA or pPZP-Hph-NrFtrA was performed as described elsewhere (Shao et al, 2015 ).…”

Section: Methodsmentioning

confidence: 99%

Siderophore Biosynthesis but Not Reductive Iron Assimilation Is Essential for the Dimorphic Fungus Nomuraea rileyi Conidiation, Dimorphism Transition, Resistance to Oxidative Stress, Pigmented Microsclerotium Formation, and Virulence

Wang

Liu

et al. 2016

Front. Microbiol.

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Iron is an indispensable factor for the dimorphic insect pathogenic Nomuraea rileyi to form persistent microsclerotia which can replace conidia or blastospores for commercial mass production. There are two high affinity iron acquisition pathways in N. rileyi, siderophore-assisted iron mobilization and reductive iron assimilation systems. Transcription of the two iron uptake pathways related genes is induced under iron-limiting conditions. Stage-specific iron uptake-related genes expression during microsclerotia development shows siderophore-mediated iron acquisition genes are rigorously upregulated specifically during the formation and mature period while reductive iron assimilation related genes just display a higher expression at the late maturation period. Abrogation of reductive iron assimilation, by the deletion of the high affinity iron permease (NrFtrA), has no visible effect on microsclerotia biogenesis in N. rileyi. In sharp contrast, N. rileyi L-ornithine-N5-monooxygenase (NrSidA), required for synthesis of all siderophores, is absolutely necessary for the development of pigmented microsclerotia. In agreement with the lower intracellular iron contents of microsclerotia in ΔNrSidA strains, not only the pigments, but both the number and the biomass are also noticeably reduced. Certain concentration of ROS is required for promoting microsclerotia biogenesis. Combined with expression pattern analysis of related genes and quantitative of intracellular iron or extracellular siderophore in WT and mutants, these data demonstrate the lack of adequate intracellular iron caused by the loss of the siderophore results in the deficiency of ROS detoxication. Furthermore, ΔNrSidA strains show significantly increased sensitivity to hydrogen peroxide. Besides, NrSidA, but not NrFtrA, play a crucial role in vegetative growth under iron-limiting conditions, conidiation, and dimorphic switching. Remarkably, the slower growth of the ΔNrSidA strains in vivo due to a reduced capacity for iron acquisition leads to the loss of virulence in Spodoptera litura while the ΔNrFtrA mutants behaved as WT during infection. Together, these results prove siderophore-assisted iron mobilization is the major pathway of cellular iron uptake and essential for conidiation, dimorphism transition, oxidative stress resistance, pigmented microsclerotium formation and full virulence.

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Agrobacterium tumefaciens -mediated transformation of the entomopathogenic fungus Nomuraea rileyi

Cited by 21 publications

References 36 publications

The high osmotic response and cell wall integrity pathways cooperate to regulate morphology, microsclerotia development, and virulence in Metarhizium rileyi

The high osmotic response and cell wall integrity pathways cooperate to regulate morphology, microsclerotia development, and virulence in Metarhizium rileyi

A transcription factor, MrMsn2, in the dimorphic fungus Metarhizium rileyi is essential for dimorphism transition, aggravated pigmentation, conidiation and microsclerotia formation

Siderophore Biosynthesis but Not Reductive Iron Assimilation Is Essential for the Dimorphic Fungus Nomuraea rileyi Conidiation, Dimorphism Transition, Resistance to Oxidative Stress, Pigmented Microsclerotium Formation, and Virulence

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