Zhimin Hao

StSTE12 is required for the pathogenicity of Setosphaeria turcica by regulating appressorium development and penetration.

In filamentous fungi, the pathogenic mitogen-activated protein kinase (PMK) pathway performs an important function in plant infection. STE12-like genes found in higher eukaryotes encode transcription factors and are regulated by the PMK pathway. However, the functions of STE12-like genes in foliar pathogens remain poorly understood. In this study, we cloned StSTE12 from Setosphaeria turcica and investigated its functions by RNA interference. Transformants ste12-3, ste12-2 and, ste12-1, in which the StSTE12 silencing efficiency increased in order, were confirmed by real time PCR. Compared with the wild-type (WT) strain, the transformants showed reduced growth rate, lighter colony color, and obviously decreased conidium production. More importantly, different to WT strain and ste12-3 with lower StSTE12silencing efficiency, ste12-1 and ste12-2 with higher StSTE12 silencing efficiency were nonpathogenic on intact leaves, but pathogenic on wounded leaves. However, the biological activity of HT-toxin from all transformants showed no difference on corn leaves. Furthermore, ste12-1 and ste12-2 did not penetrate artificial cellophane membrane and showed abnormal and delayed development appressoria. Although it could penetrate the cellophane membranes, ste12-3 formed appressoria after 48 h of inoculation more than WT. Therefore, StSTE12 was involved in vegetative growth, conidiation, appressorial development, penetration as well as the pathogenicity, but it was not related to HT-toxin biosynthesis. More interestingly, all the results suggested that StSTE12 was crucial for pathogenicity due to involvement in regulating appressoria development and penetration.

The StLAC2 gene is required for cell wall integrity, DHN-melanin synthesis and the pathogenicity of Setosphaeria turcica.

Laccases are blue multicopper oxidases, play important roles in various biological processes. These processes include fungal dihydroxynaphthalene (DHN)-melanin biosynthesis and pathogenicity, cellular growth, morphogenesis, and differentiation. This study investigated functions of the laccase gene StLAC2 in Setosphaeria turcica. The Δlac2 mutant colony color was distinct from that of the S. turcica wild-type (WT) isolate, and the mutants exhibited defective conidial formation. In contrast to the WT, the mutants exhibited a lighter color on the 2, 2-azino-di-[3-ethylbenzo-thia-zolin-sulphonate] (ABTS) plates, and the intracellular laccase activity was lower. Notably, StLAC2 gene loss correlated with decreased DHN-melanin biosynthesis and affected the integrity of the cell wall, where the StLAC2 gene mutants showed thinner, more transparent walls with a higher number of mitochondria than the WT. The Δlac2 mutants also lost their pathogenicity in maize. The results indicated that the StLAC2 gene involved in cell wall integrity, melanin biosynthesis and appressorial and conidial formation.

MAP kinase gene STK1 is required for hyphal, conidial, and appressorial development, toxin biosynthesis, pathogenicity, and hypertonic stress response in the plant pathogenic fungus Setosphaeria turcica

Abstract The mitogen-activated protein kinase (MAPK), a key signal transduction component in the MAPK cascade pathway, regulates a variety of physiological activities in eukaryotes. However, little is known of the role MAPK plays in phytopathogenic fungi. In this research, we cloned the MAPK gene STK1 from the northern corn leaf blight pathogen Setosphaeria turcica and found that the gene shared high homology with the high osmolality glycerol (HOG) MAPK gene HOG1 of Saccharomyces cerevisiae. In addition, gene knockout technology was employed to investigate the function of STK1 . Gene knockout mutants (KOs) were found to have altered hyphae morphology and no conidiogenesis, though they did show similar radial growth rate compared to the wild-type strain (WT). Furthermore, microscope observations indicated that STK1 KOs did not form normal appressoria at 48 h post-inoculation on a hydrophobic surface. STK1 KOs had reduced virulence, a significantly altered Helminthosporium turcicum (HT)-toxin composition, and diminished pathogenicity on the leaves of susceptible inbred corn OH43. Mycelium morphology appeared to be significantly swollen and the radial growth rates of STK1 KOs declined in comparison with WT under high osmotic stress. These results suggested that STK1 affects the hyphae development, conidiogenesis, and pathogenicity of S. turcica by regulating appressorium development and HT-toxin biosynthesis. Moreover, the gene appears to be involved in the hypertonic stress response in S. turcica.

Efficient strategy for introducing large and multiple changes in plasmid DNA

While the QuikChange site-directed mutagenesis method and its later modifications are extremely useful and simple, they suffer from several drawbacks. Here, we propose a new method, named LFEAP mutagenesis (Ligation of Fragment Ends After PCR) for creating various mutations in plasmid by leveraging three existing concepts: inverse PCR, single primer PCR, and sticky-end assembly. The first inverse PCR on the target plasmid yielded linearized DNA fragments with mutagenic ends, and a second single primer PCR resulted in complementary single-stranded DNA fragments with the addition of overhangs at the 5′ end of each strand. The resulting single strands were then annealed to produce double-stranded DNA with free 5′ single-stranded DNA tails. These products with compatible sticky ends were efficiently assembled into a circular, mutagenized plasmid. With this strategy, multiple simultaneous changes (up to 15) and mutations in large plasmids (up to 50 kb) were achieved with high efficiency and fidelity. LFEAP mutagenesis is a versatile method that offers significant advantages for introducing large and multiple changes in plasmid DNA.

A new strategy for seamless gene editing and marker recycling in Saccharomyces cerevisiae using lethal effect of Cwp1

Technologies development for seamless gene editing and marker recycling has allowed frequent genomic engineering in Saccharomyces cerevisiae for desired laboratory strains and cell factory. Alternative new approaches are still required for complicated scenarios. In this study, we report that inducible overexpression of cell wall protein 1 (Cwp1) by galactose addition confers yeast cells a robust growth inhibition. Direct repeats flanking the Gal‐CWP1:selectable marker cassette allow for its homology recombination excision and counter selection upon galactose addition, therefore enable seamless gene editing and marker recycling. We used this strategy and efficiently generated scarless Ade8 deletion mutants. Our results highlight the utility of lethal effect of Cwp1 overexpression a new counter selection strategy and a simple and efficient method for seamless gene editing and marker recycling in S. cerevisiae and potentially other fungi.

The heterotrimeric G protein г Stgg1 is required for conidiation, secondary metabolite production and pathogenicity of Setosphaeria turcica

ABSTRACT Heterotrimeric G proteins are best known for their role in the transduction of extracellular signals to various downstream effectors. G proteins in higher eukaryotes are intensively studied; however, their roles in foliar pathogens are still elusive. In this study, we cloned the gene Stgg1 encoding G protein γ subunit in Setosphaeria turcica and investigated its function by RNA interference technology. Three independent Stgg1 targeted RNAi mutants R3, R5 and R6 with diverse silencing efficiency were generated. Knock-down of Stgg1 resulted in a significant reduction in mRNA levels of the genes encoding Gα (Stga1, Stga2, Stga3) but not for Gβ (Stgb1). Stgg1 RNAi mutants exhibited significantly elongated hyphal cells with blocked conidium production. In addition, Stgg1 RNAi mutants all appeared in lighter colony colour compatible with inhibited secondary metabolites. Further assays demonstrated that Stgg1 was required for biosynthesis of melanin and HT-toxin activity. Furthermore, down-regulation of Stgg1 largely inhibited the inflection capacity. Thus, we proposed that Stgg1 played crucial roles in conidiation, secondary metabolite production and pathogenicity of S. turcica and is, therefore, an ideal target for drug design against foliar pathogens.

StPBS2, a MAPK kinase gene, is involved in determining hyphal morphology, cell wall development, hypertonic stress reaction as well as the production of secondary metabolites in Northern Corn Leaf Blight pathogen Setosphaeria turcica

Mitogen activated protein kinase kinase (MAPKK) is a crucial component in the MAPK signaling pathway. However, the functions of MAPKKs in foliar pathogens remain poorly understood. In the current study, a MAPKK gene designated as StPBS2 was cloned from Setosphaeria turcica and the functions of this gene were investigated by RNAi technology. Four independent StPBS2 gene silence transformants with different efficiencies were confirmed by real time PCR. Compared to the wild type strain (WT), these transformants showed decreased colony growth, shortened hyphae cell length, broadened cell width and an obvious reduction in conidium yield. Moreover, the cell wall of the transformants was thicker and they were also more sensitive to substances that interfere with cell wall biosynthesis than WT. Additionally, the transformants displayed higher sensitivity to hypertonic stress than WT and the sensitivity was associated with the level of silencing of StPBS2. They were also resistant to the fungicides iprodione, procymidone and fludioxonil, to which WT almost completely sensitive. The transformants produced more red secondary metabolites than WT and the production was enhanced with increasing silencing level and increased glucose content in PDA medium. Our results suggest that StPBS2 is involved in morphogenesis, condiogenesis, cell wall development, hypertonic stress reaction and resistance to fungicides, as well as in the biosynthesis of secondary metabolites in S. turcica.