The fungal pathogen Setosphaeria turcica causes northern corn leaf blight (NCLB), which leads to considerable crop losses.  Setosphaeria turcica elaborates a specialized infection structures called appressorium for maize infection.  Previously, we demonstrated that the S. turcica triggers an S-phase checkpoint and ATR (Ataxia Telangiectasia and Rad3 related)-dependent self-protective response to DNA genotoxic insults during maize infection.  However, how the regulatory mechanism works was still largely unknown.  Here, we report a genome wide transcriptional profile analysis during appressorium formation in the present of DNA replication stress.  We performed RNA-Seq analysis to identify S. tuicica genes responsive to DNA replication stress.  In the current work, we found that appressorium-mediated maize infection by S. turcica is significantly blocked by S-phase checkpoint.  A large serial of secondary metabolite and melanin biosynthesis genes were blocked in appressorium formation of S. turcica during the replication stress.  The secondary metabolite biosynthesis genes including alcohol dehydrogenase GroES-like domain, multicopper oxidase, ABC-transporter families, cytochrome P450 and FAD-containing monooxygenase were related to plant pathogen infection.  In addition, we demonstrated that autophagy in S. turcica is up-regulated by ATR as a defense response to stress.  We identified StATG3, StATG4, StATG5, StATG7 and StATG16 genes for autophagy were induced by ATR-mediated S-phase checkpoint.  We therefore propose that in response to genotoxic stress, S. turcica utilizes ATR-dependent pathway to turn off transcription of genes governing appressorium-mediated infection, and meanwhile inducing transcription of autophagy genes likely as a mechanism of self-protection, aside from the more conservative responses in eukaryotes.

Laccases, as a kind of multicopper oxidase, play an important role in pigment synthesis and growth in fungi and are involved in their interactions with host plants.  In Setosphaeria turcica, 9 laccase-like multicopper oxidases have been identified, and StLAC2 is involved in the synthesis of the melanin that accumulates in the cell wall.  The function of another major laccase gene, StLAC6, was studied here.  The knockout of StLAC6 had no effect on the growth, morphology or invasion ability of S. turcica, but the morphology and function of peroxisomes of knockout mutants were abnormal.  The knockout of the StLAC6 gene resulted in increased contents of phenolic compounds and melanin and the sensitivity to fungicides increased compared with wild type strains.  In the mutants of StLAC6, there is a significant change of the expression levels of other laccase genes.  This study provides a new insight into laccase functions and the relationship of the laccase gene family in plant pathogenic fungi.   

Setosphaeria turcica (syn. Exserohilum turcicum) is the pathogenic fungus of maize (Zea mays) that causes northern leaf blight, which is a major maize disease worldwide.  Melanized appressoria are highly specialized infection structures formed by germinated conidia of S. turcica that infect maize leaves.  The appressorium penetrates the plant cuticle by generating turgor, and glycerol is known to be the main source of the turgor.  Here, the infection position penetrated by the appressorium on maize leaves was investigated, most of the germinated conidia entered the leaf interior by directly penetrating the epidermal cells, and the appressorium structure was necessary for the infection, whether it occurred through epidermal cells or stomata.  Then, to investigate the effects of key factors in the development of the appressorium, we studied the effects of three inhibitors, including a melanin inhibitor (tricyclazole, TCZ), a DNA replication inhibitor (hydroxyurea, HU), and an autophagy inhibitor (3-methyladenine, 3-MA), on appressorium turgor and glycerol content.  As results, appressorium turgor pressure and glycerol concentration in the appressorium reached their highest levels at the mature stage of the appressorium under the control and inhibitor treatments.  The three inhibitors had the greatest effects on appressorium turgor pressure at this stage.  Glycogen and liposomes are the main substances producing glycerol.  It was also found inhibitors affected the distribution of glycogen and liposomes, which were detected in the conidia, the germ tube, and the appressorium during appressorium development.  This study provides profound insight into the relationship between appressorium turgor pressure and glycerol content, which was affected by the synthesis of melanin, DNA replication, and autophagy in the developing appressorium during a S. turcica infection.  

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.

Natural herbicides, or environment-friendly bioherbicides have been attracted more and more attentions. Isolation and structural identification of natural herbicide-active compounds from plant pathogens has been proved to be an effective approach for novel lead discovery of the pesticide development. In this study, the metabolites of the mutant strain PAM1, which obtained from PA1 of Pythium aphanidermatum (Eds.) Fitzp by ultraviolet radiation were separated and identified by HPLC, NMR, and IR. The results revealed that three active compounds including 4-hydroxy-3-methoxycinnamic acid and two indole derivatives, exhibited inhibition activity on the elongation of radical and coleoptile of Digtaria sanguinalis (L.) Scop.