Conditional gene expression and promoter replacement in  Zymoseptoria tritici  using fungal nitrate reductase promoters

Marchegiani, Elisabetta; Sidhu, Yaadwinder; Haynes, Ken; Lebrun, Marc

doi:10.1016/j.fgb.2015.04.021

Cited by 13 publications

(10 citation statements)

References 31 publications

(40 reference statements)

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“…Sequence analysis revealed that FgNeo1 is a putative homolog of yeast Neo1, a phospholipid-translocating ATPase (Ca 2+ ATPase) that is essential in yeast [46]. Therefore, we adopted the conditional promoter replacement (CPR) approach for inducing (NaNO 3 : NO 3 - ) or silencing (Glutamate: Glu) FgNEO1 under the control of pFgNIA1 [47] to characterize its function. Three CPR transformants ( pFgNIA1-FgNEO1 ) were identified by PCR screening (S4A and S4B Fig) and verified by qRT-PCR analysis (Fig 8A).…”

Section: Resultsmentioning

confidence: 99%

The t-SNARE protein FgPep12, associated with FgVam7, is essential for ascospore discharge and plant infection by trafficking Ca2+ ATPase FgNeo1 between Golgi and endosome/vacuole in Fusarium graminearum

Dong

Zhao

et al. 2019

PLoS Pathog

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Soluble N-ethylmaleimide-sensitive factor attachment receptors (SNAREs) play a crucial role in the development and virulence through mediation of membrane fusion and vesicle trafficking in pathogens. Our previous studies reported that the SNARE protein FgVam7 and its binding proteins FgVps39/41 are involved in vesicle trafficking and are important for vegetative growth, asexual/sexual development, deoxynivalenol production and virulence in the Fusarium head blight fungus Fusarium graminearum . Here, we identified and characterized another FgVam7 binding protein in F . graminearum , FgPep12, an ortholog of yeast t-SNARE Pep12 with both the SNARE and TM domains being essential for its localization and function. Deletion of FgPep12 caused defects in vegetative growth, conidiogenesis, deoxynivalenol production and virulence. Cytological observation revealed that FgPep12 localizes to the Golgi apparatus, late endosomes and vacuoles, and is necessary for transport from the vacuole to prevacuolar compartment. Further investigation revealed that both FgPep12 and FgVam7 are essential for ascospore discharge through interaction with and trafficking of the Ca 2+ ATPase FgNeo1 between the Golgi and endosomal/vacuolar system. FgNeo1 has similar biological roles to FgPep12 and is required for ascospore discharge in F . graminearum . Together, these results provide solid evidence to help unravel the mechanisms underlying the manipulation of ascospore discharge and plant infection by SNARE proteins in F . graminearum .

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Section: Resultsmentioning

confidence: 99%

The t-SNARE protein FgPep12, associated with FgVam7, is essential for ascospore discharge and plant infection by trafficking Ca2+ ATPase FgNeo1 between Golgi and endosome/vacuole in Fusarium graminearum

Dong

Zhao

et al. 2019

PLoS Pathog

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“…The Stb6 -avirulent IPO323 isolate (avirulent on Stb16q ), collected on cultivar “Arminda” in the Netherlands in 1981, is the genome reference isolate ( Goodwin et al, 2011 ). For cytological analyses, plasmid pYSKH-4 was introduced into Z. tritici isolates IPO9415 and CFZ008 using Agrobacterium tumefasciens -mediated transformation (ATMT) according to published protocols ( Marchegiani et al, 2015 ). Plasmid pYSKH-4 contains eGFP expressed under the control of a strong constitutive promoter TEF1 and the ILV1-R gene, conferring resistance to sulfonylurea ( Sidhu et al, 2015 ).…”

Section: Methodsmentioning

confidence: 99%

Blocked at the Stomatal Gate, a Key Step of Wheat Stb16q-Mediated Resistance to Zymoseptoria tritici

et al. 2022

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Septoria tritici blotch (STB), caused by the fungus Zymoseptoria tritici, is among the most threatening wheat diseases in Europe. Genetic resistance remains one of the main environmentally sustainable strategies to efficiently control STB. However, the molecular and physiological mechanisms underlying resistance are still unknown, limiting the implementation of knowledge-driven management strategies. Among the 22 known major resistance genes (Stb), the recently cloned Stb16q gene encodes a cysteine-rich receptor-like kinase conferring a full broad-spectrum resistance against Z. tritici. Here, we showed that an avirulent Z. tritici inoculated on Stb16q quasi near isogenic lines (NILs) either by infiltration into leaf tissues or by brush inoculation of wounded tissues partially bypasses Stb16q-mediated resistance. To understand this bypass, we monitored the infection of GFP-labeled avirulent and virulent isolates on Stb16q NILs, from germination to pycnidia formation. This quantitative cytological analysis revealed that 95% of the penetration attempts were unsuccessful in the Stb16q incompatible interaction, while almost all succeeded in compatible interactions. Infectious hyphae resulting from the few successful penetration events in the Stb16q incompatible interaction were arrested in the sub-stomatal cavity of the primary-infected stomata. These results indicate that Stb16q-mediated resistance mainly blocks the avirulent isolate during its stomatal penetration into wheat tissue. Analyses of stomatal aperture of the Stb16q NILs during infection revealed that Stb16q triggers a temporary stomatal closure in response to an avirulent isolate. Finally, we showed that infiltrating avirulent isolates into leaves of the Stb6 and Stb9 NILs also partially bypasses resistances, suggesting that arrest during stomatal penetration might be a common major mechanism for Stb-mediated resistances.

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“…To circumvent this, we introduced the nitrate reductase MoNIA1 promoter, a conditional promoter described previously in studies of Z. tritici and Aspergillus fumigatus (42, 43), into MoARF1 and MoSAR1 . We obtained one Δ Moarf1 / CPR mutant and one Δ Mosar1 / CPR mutant, confirmed by Southern blotting (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…tritici and A. fumigatus (42, 43). Two approximately 1.0-kb DNA fragments flanking the promoter of MoARF1 or MoSAR1 and the promoter of MoNIA were amplified using the primer pairs (Table S2).…”

Section: Methodsmentioning

confidence: 99%

System-Wide Characterization of MoArf GTPase Family Proteins and Adaptor Protein MoGga1 Involved in the Development and Pathogenicity of Magnaporthe oryzae

Zhang

Yang

et al. 2019

mBio

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ADP ribosylation factor (Arf) small GTPase family members are involved in vesicle trafficking and organelle maintenance in organisms ranging from Saccharomyces cerevisiae to humans. A previous study identified Magnaporthe oryzae Arf6 (MoArf6) as one of the Arf proteins that regulates growth and conidiation in the rice blast fungus M. oryzae, but the remaining family proteins remain unknown. Here, we identified six additional Arf proteins, including MoArf1, MoArl1, MoArl3, MoArl8, MoCin4, and MoSar1, as well as their sole adaptor protein, MoGga1, and determined their shared and specific functions. We showed that the majority of these proteins exhibit positive regulatory functions, most notably, in growth. Importantly, MoArl1, MoCin4, and MoGga1 are involved in pathogenicity through the regulation of host penetration and invasive hyphal growth. MoArl1 and MoCin4 also regulate normal vesicle trafficking, and MoCin4 further controls the formation of the biotrophic interfacial complex (BIC). Moreover, we showed that Golgi-cytoplasm cycling of MoArl1 is required for its function. Finally, we demonstrated that interactions between MoArf1 and MoArl1 with MoGga1 are important for Golgi localization and pathogenicity. Collectively, our findings revealed the shared and specific functions of Arf family members in M. oryzae and shed light on how these proteins function through conserved mechanisms to govern growth, transport, and virulence of the blast fungus. IMPORTANCE Magnaporthe oryzae is the causal agent of rice blast, representing the most devastating diseases of rice worldwide, which results in losses of amounts of rice that could feed more than 60 million people each year. Arf (ADP ribosylation factor) small GTPase family proteins are involved in vesicle trafficking and organelle maintenance in eukaryotic cells. To investigate the function of Arf family proteins in M. oryzae, we systematically characterized all seven Arf proteins and found that they have shared and specific functions in governing the growth, development, and pathogenicity of the blast fungus. We have also identified the pathogenicity-related protein MoGga1 as the common adaptor of MoArf1 and MoArl1. Our findings are important because they provide the first comprehensive characterization of the Arf GTPase family proteins and their adaptor protein MoGga1 functioning in a plant-pathogenic fungus, which could help to reveal new fungicide targets to control this devastating disease.

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Conditional gene expression and promoter replacement in Zymoseptoria tritici using fungal nitrate reductase promoters

Cited by 13 publications

References 31 publications

The t-SNARE protein FgPep12, associated with FgVam7, is essential for ascospore discharge and plant infection by trafficking Ca2+ ATPase FgNeo1 between Golgi and endosome/vacuole in Fusarium graminearum

The t-SNARE protein FgPep12, associated with FgVam7, is essential for ascospore discharge and plant infection by trafficking Ca2+ ATPase FgNeo1 between Golgi and endosome/vacuole in Fusarium graminearum

Blocked at the Stomatal Gate, a Key Step of Wheat Stb16q-Mediated Resistance to Zymoseptoria tritici

System-Wide Characterization of MoArf GTPase Family Proteins and Adaptor Protein MoGga1 Involved in the Development and Pathogenicity of Magnaporthe oryzae

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