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

Electrostatic interactions between the A-subunit and LCMT-1.

<p>(<b>A</b>) Structural model of the PP2A core enzyme-LCMT-1 complex built from the structure of the PP2Ac-LCMT-1 complex and the A-subunit with morphed conformation. The electrostatic potential for the A-subunit (left panel) and LCMT-1 (right panel) is shown. The corresponding regions on the A-subunit and LCMT-1 for potential electrostatic interactions are circled (i and ii). The A-subunit residues that directly contact LCMT-1 in the model, Arg183 and Arg258, are shown. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086955#pone.0086955.s003" target="_blank">Movie S1</a>. (<b>B</b>) The effect of the A-subunit mutations to Arg183 and Arg258 on methylation of the PP2A core enzyme. Results from 5–7 independent experiments were summarized and the average K_m ± std. dev. and the P value were calculated and shown. <b>(C)</b> FRET assay measured changes in the distance between the N- and C-termini of the A-subunit in the core enzyme prior to and after addition of an excess molar amount of LCMT-1 or a stoichiometric amount of PR70 (108–575). Representative results were shown with mean ± SEM calculated from triplicate.</p

Mechanisms of the Scaffold Subunit in Facilitating Protein Phosphatase 2A Methylation

<div><p>The function of the biologically essential protein phosphatase 2A (PP2A) relies on formation of diverse heterotrimeric holoenzymes, which involves stable association between PP2A scaffold (A) and catalytic (C or PP2Ac) subunits and binding of variable regulatory subunits. Holoenzyme assembly is highly regulated by carboxyl methylation of PP2Ac-tail; methylation of PP2Ac and association of the A and C subunits are coupled to activation of PP2Ac. Here we showed that PP2A-specific methyltransferase, LCMT-1, exhibits a higher activity toward the core enzyme (A–C heterodimer) than free PP2Ac, and the A-subunit facilitates PP2A methylation via three distinct mechanisms: 1) stabilization of a proper protein fold and an active conformation of PP2Ac; 2) limiting the space of PP2Ac-tail movement for enhanced entry into the LCMT-1 active site; and 3) weak electrostatic interactions between LCMT-1 and the N-terminal HEAT repeats of the A-subunit. Our results revealed a new function and novel mechanisms of the A-subunit in PP2A methylation, and coherent control of PP2A activity, methylation, and holoenzyme assembly.</p></div

The effect of the N-terminal HEAT repeats of the A-subunit on methylation.

<p>(<b>A</b>) Schematic representation of internal truncations of the N-terminal HEAT repeats to reduce the length of the A-subunit N-terminal structure. (<b>B</b>) Normalized concentrations of PP2Ac assembled with full-length or truncated A-subunits used for kinetic analysis of PP2A methylation in (<b>C</b>). (<b>C</b>) Kinetics of methylation of PP2A core enzymes with varied length of the A-subunit. Results from 6–7 independent experiments were summarized and the average K_m ± std. dev. was calculated and shown in the table on the right. (<b>D</b>) Correlation between the number of deleted N-terminal HEAT repeats and the K_m of methylation of PP2A core enzyme by LCMT-1. Statistical test of this correlation was performed and the P value was calculated and shown. (<b>E</b>) Structure of the core enzyme portion of PP2A holoenzyme (PDB ID: 2NPP). The A-subunit, PP2Ac, and PP2Ac-tail are colored green, blue, and magenta, respectively. The positions of the HEAT-repeats where truncations were made are indicated by numbers.</p

The A-subunit enhances PP2A methylation and interaction with LCMT-1.

<p>(<b>A</b>) Influence of the A-subunit, mini-A, and PTPA on PP2A methylation, tested by addition of these proteins in a stoichiometric amount to PP2Ac, prior to determination of the kinetics of PP2A methylation. Representative results were shown with mean ± SEM calculated from triplicate (upper figure panel). Statistical analysis of 4–12 independent experiments was performed and the average K_m ± std. dev. and the P value were calculated and summarized in the table below. (<b>B</b>) Binding affinity between LCMT-1, preloaded with SAH, and unmethylated, methylated PP2A core enzyme or free PP2Ac, measured by ITC.</p

Shortening of PP2Ac-tail enhanced methylation.

<p>(<b>A</b>) Schematic representation of Δ294–298 truncation of PP2Ac and the range of PP2Ac-tail mobility. PP2Ac is shown as blue sphere with two small red spheres representing catalytic metal ions. The PP2Ac-tail is shown in cyan. The cyan spheres surrounding PP2Ac indicate the range of movement of PP2Ac-tail. Note that the Δ294–298 truncation reduces the space for the PP2Ac-tail movement. (<b>B</b>) Kinetics of methylation of PP2Ac Δ294–298 and full-length PP2Ac either as the free catalytic subunit or as the core enzymes assembled with the A-subunit with internal deletion of increasing number of N-terminal HEAT repeats. Results from 3–9 independent experiments were summarized and the average K_m ± std. dev. and the P value were calculated and shown. (<b>C</b>) Kinetics of methylation of PP2Ac Δ294–298 associated with the A-subunit with internal deletion of increasing number of N-terminal HEAT repeats (left) and comparison of the full-length PP2Ac and PP2Ac Δ294–298 in correlation of the K_m of methylation and the number of N-terminal HEAT repeats of the A-subunit deleted in the core enzymes (right). Statistical test of this correlation was performed and P values were calculated and shown.</p

Summary of original sequencing data.

<p>Summary of original sequencing data.</p

large-effect SNPs between samples.

<p>large-effect SNPs between samples.</p

Comparative distributions of variation frequency on 12 chromosomes.

<p>s1, IR24; S2, MH63; S3,SH527.</p

Syn_CDS and Non_syn_CDS SNPs variations between samples.

<p>Syn_CDS and Non_syn_CDS SNPs variations between samples.</p