Polarity proteins Mrcdc24 and Mrbem1 required for hypha growth and microsclerotia formation in <i>Metarhizium rileyi</i>

<p>Polarity proteins cdc24 and bem1 regulate polar growth, cytoskeleton morphogenesis, and the generation of reactive oxygen species (ROS) in filamentous fungi. In this study, the <i>Mrcdc24</i> and <i>Mrbem1</i> genes were cloned from <i>Metarhizium rileyi</i> and found to encode 1006 and 562 amino acid proteins, respectively. To determine the functions of <i>Mrcdc24</i> and <i>Mrbem1</i>, gene-silencing mutants <i>Mrcdc24RNAi</i>, <i>Mrbem1RNAi</i>, and <i>Mrcdc24&Nrbem1RNAi</i> were generated by RNA silencing. The mutants presented significant phenotype changes in hyphal growth, and conidial yields, microsclerotia yields and virulence were reduced. Furthermore, the transcription levels of the <i>MrnoxA</i> and <i>MrnoxR,</i> which regulate ROS generation in the <i>Mrcdc24RNAi and Mrbem1RNAi</i> strains, were reduced. The transcriptional levels of <i>MrracA</i> and <i>Mrcdc42</i> were significantly reduced in the <i>Mrcdc24RNAi</i> strains. Our results confirmed the crucial role of genes <i>Mrcdc24</i> and <i>Mrbem1</i> in hyphal growth, conidiation, microsclerotia formation, ROS generation and virulence.</p

Length distribution of assembled unigenes.

<p>The number of y-axis has been transfer into logarithmic scale.</p

Homology search of Illumina sequences against the Nr database.

<p><b>(A)</b> E-value distribution of BLAST hits for each unique sequence with a cut-off E-value of 1.0E^-5. <b>(B)</b> Species distribution of the top BLAST hits for each sequence. <b>(C)</b> Species distribution as a percentage of the total homologous sequences with an E-value ≥1.0E^-5. The first hit of each sequence was used for analysis.</p

Gene Ontology analysis of genes in the Illumina <i>de novo</i> transcriptome of <i>M</i>. <i>cichorii</i>.

<p>The results are summarized in three main categories: biological process, cellular component, and molecular function. The righty-axis indicates the number of genes in a category. The left y-axis indicates the percentage of a specific category of genes in that main category.</p

COG functional annotation of putative proteins.

<p>Of 23,739 Nr hits, 8,580 sequences had COG classifications among the 25 categories. The y-axis indicates the number of sequences in a category.</p

“Terpenoid backbone biosynthesis” pathway of <i>M</i>. <i>cichorii de novo</i> transcriptome.

<p>The red box indicates the homologous enzyme of the unigenes have been found in the transcriptome databases of <i>M</i>. <i>cichorii</i>, while the white box represents there is no unigene has been found.</p

RT-qPCR analysis of 13 unigenes differentially-expressed between 20–25 day-old female and male groups.

<p>Line diagrams represent the expression patterns of selected unigenes from the <i>M</i>. <i>cichorii</i> transcriptome. Expression was compared between female and male groups. Error bars represent standard error. *Significant difference between gene expression (based on 20–25 day-old females, using Student’s <i>t</i>-test with p < 0.05).</p