Global Protein–Protein Interaction Network of Rice Sheath Blight Pathogen

<i>Rhizoctonia solani</i> is the major pathogenic fungi of rice sheath blight. It is responsible for the most serious disease of rice (<i>Oryza sativa</i> L.) and causes significant yield losses in rice-growing countries. Identifying the protein–protein interaction (PPI) maps of <i>R. solani</i> can provide insights into the potential pathogenic mechanisms and assign putative functions to unknown genes. Here, we exploited a PPI map of <i>R. solani</i> anastomosis group 1 IA (AG-1 IA) based on the interolog and domain–domain interaction methods. We constructed a core subset of high-confidence protein networks consisting of 6705 interactions among 1773 proteins. The high quality of the network was revealed by comprehensive methods, including yeast two-hybrid experiments. Pathogenic interaction subnetwork, secreted proteins subnetwork, and mitogen-activated protein kinase (MAPK) cascade subnetwork and their interacting partners were constructed and analyzed. Moreover, to exactly predict the pathogenic factors, the expression levels of the interaction proteins were investigated by analyzing RNA sequences that consisted of samples from the entire infection progress. The PPIs offer an exceptionally rich source of data that can be used to understand the gene functions and biological processes of this serious disease at the system level

Biosynthesis of Antibiotic Leucinostatins in Bio-control Fungus <i>Purpureocillium lilacinum</i> and Their Inhibition on <i>Phytophthora</i> Revealed by Genome Mining

<div><p><i>Purpureocillium lilacinum</i> of Ophiocordycipitaceae is one of the most promising and commercialized agents for controlling plant parasitic nematodes, as well as other insects and plant pathogens. However, how the fungus functions at the molecular level remains unknown. Here, we sequenced two isolates (PLBJ-1 and PLFJ-1) of <i>P</i>. <i>lilacinum</i> from different places Beijing and Fujian. Genomic analysis showed high synteny of the two isolates, and the phylogenetic analysis indicated they were most related to the insect pathogen <i>Tolypocladium inflatum</i>. A comparison with other species revealed that this fungus was enriched in carbohydrate-active enzymes (CAZymes), proteases and pathogenesis related genes. Whole genome search revealed a rich repertoire of secondary metabolites (SMs) encoding genes. The non-ribosomal peptide synthetase LcsA, which is comprised of ten C-A-PCP modules, was identified as the core biosynthetic gene of lipopeptide leucinostatins, which was specific to <i>P</i>. <i>lilacinum</i> and <i>T</i>. <i>ophioglossoides</i>, as confirmed by phylogenetic analysis. Furthermore, gene expression level was analyzed when PLBJ-1 was grown in leucinostatin-inducing and non-inducing medium, and 20 genes involved in the biosynthesis of leucionostatins were identified. Disruption mutants allowed us to propose a putative biosynthetic pathway of leucinostatin A. Moreover, overexpression of the transcription factor <i>lcsF</i> increased the production (1.5-fold) of leucinostatins A and B compared to wild type. Bioassays explored a new bioactivity of leucinostatins and <i>P</i>. <i>lilacinum</i>: inhibiting the growth of <i>Phytophthora infestans</i> and <i>P</i>. <i>capsici</i>. These results contribute to our understanding of the biosynthetic mechanism of leucinostatins and may allow us to utilize <i>P</i>. <i>lilacinum</i> better as bio-control agent.</p></div

Lifestyles of nematophagous <i>P</i>. <i>lilacinum</i> and the structures of leucinostatins.

<p>(A) Microscopic conidiophores and conidia (c) of <i>P</i>. <i>lilacinum</i>. Scale bar = 10 μm. The soil saprophyte (s) <i>P</i>. <i>lilacinum</i> colonizes plant roots as an endophyte (e), and the parasite (p) can occur in nematode eggs in the egg mass (em) generated after the infection with the plant nematode (n). (B) Chemical structure of leucinostatins A and B.</p

Genome feature of the three <i>P</i>. <i>lilacinum</i> isolates.

<p>Genome feature of the three <i>P</i>. <i>lilacinum</i> isolates.</p

The boundary of the <i>lcs</i> cluster in <i>P</i>. <i>lilacinum</i> with its homologues in <i>T</i>. <i>ophioglossoides</i>.

<p>(A) Horizontal arrows of the same color represent the orthologous genes. The sequence identity between the homologous genes from two fungi is shown by shaded areas with different colors. TO, <i>T</i>. <i>ophioglossoides</i>; PL, <i>P</i>. <i>lilacinum</i>. The bars indicate boundaries of the <i>lcs</i> cluster predicted by antiSMASH, SMURF, and qRT-PCR. (B) The expression ratio of the genes around <i>lcsA</i> when expression in PLBJ-1 cultured in leucinostatin-inducing medium was compared to expression in non-inducing medium. The ratios for different genes demonstrated an extensive range, so the breakpoint was inserted into the Y axis.</p

Description of the genes in the leucinostatin biosynthetic cluster.

<p>Description of the genes in the leucinostatin biosynthetic cluster.</p

A putative biosynthetic pathway for leucinostatin A.

<p>A putative biosynthetic pathway for leucinostatin A.</p

Phylogenomic relationships and orthologous gene clusters.

<p>(A) Maximum likelihood phylogeny was computed from a concatenated alignment of 855 groups of single-copy orthologues. Bootstrap values are shown beside the nodes. (B) The number of gene clusters shared by <i>P</i>. <i>lilacinum</i> with other major associated ecologies. Gray = <i>P</i>. <i>lilacinum</i> isolates PLBJ-1 and PLFJ-1; blue = nematode egg parasite <i>P</i>. <i>chlamydosporia</i> isolates 123 and 170; pink = nematode parasite <i>H</i>. <i>minnesotensis</i>; and yellow = nematode-trapping fungi <i>A</i>. <i>oligospora</i> and <i>M</i>. <i>haptotylum</i>.</p

Genomic synteny of PLBJ-1 and PLFJ-1.

<p>(A) The syntenic genome sequences of PLBJ-1 and PLFJ-1 were analyzed by BLASTN, with an E-value cutoff of 1e-5. The red semicircle represents the scaffolds of PLBJ-1, while the blue semicircle represents the scaffolds of PLFJ-1. Scaffold lengths of ≥ 100 Kb were used for this analysis, and the threshold of matched blocks was ≥ 1000 bp, which are connected by lines of the same color. (B) An example of a super-scaffold inferred by syntenic analysis.</p

The role of leucinostatins in antagonism between <i>P</i>. <i>lilacinum</i> and <i>Phytophthora</i>.

<p>(A) Cocultivation of <i>P</i>. <i>infestans</i> and wild type, Δ<i>lcsA</i> and OE::<i>lcsF P</i>. <i>lilacinum</i> on rye agar medium. (B) Cocultivation of <i>P</i>. <i>capsici</i> and wild type, Δ<i>lcsA</i> and OE::<i>lcsF P</i>. <i>lilacinum</i> on PDA medium.</p