Eight RGS and RGS-like proteins orchestrate growth, differentiation, and pathogenicity of Magnaporthe oryzae

Abstract

A previous study identified MoRgs1 as an RGS protein that negative regulates G-protein signaling to control developmental processes such as conidiation and appressorium formation in Magnaporthe oryzae. Here, we characterized additional seven RGS and RGS-like proteins (MoRgs2 through MoRgs8). We found that MoRgs1 and MoRgs4 positively regulate surface hydrophobicity, conidiation, and mating. Indifference to MoRgs1, MoRgs4 has a role in regulating laccase and peroxidase activities. MoRgs1, MoRgs2, MoRgs3, MoRgs4, MoRgs6, and MoRgs7 are important for germ tube growth and appressorium formation. Interestingly, MoRgs7 and MoRgs8 exhibit a unique domain structure in which the RGS domain is linked to a seven-transmembrane motif, a hallmark of G-protein coupled receptors (GPCRs). We have also shown that MoRgs1 regulates mating through negative regulation of Gα MoMagB and is involved in the maintenance of cell wall integrity. While all proteins appear to be involved in the control of intracellular cAMP levels, only MoRgs1, MoRgs3, MoRgs4, and MoRgs7 are required for full virulence. Taking together, in addition to MoRgs1 functions as a prominent RGS protein in M. oryzae, MoRgs4 and other RGS and RGS-like proteins are also involved in a complex process governing asexual/sexual development, appressorium formation, and pathogenicity.

Figure 1. M. oryzae encodes eight RGS and RGS-like proteins.

(A) Schematic representation of all eight M. oryzae RGS proteins and their comparison to those of S. cerevisiae. DEP, domains found in Dishevelled, Egl-10, and pleckstrin; PX, domains that bind to phosphoinositides; TM, transmembrane; aa, amino acids; SP, signal peptide. (B) The alignment of M. oryzae (Mo) and S. cerevisiae (Sc) RGS proteins indicates higher amino acid sequence similarity between homologs. Protein sequences were aligned, and the phylogenic tree was drawn using Clustal W 1.83. The GenBank accession numbers are as follows: MoRgs1, ABC60049; MoRgs2, XP_361183; MoRgs3, XP_360603; MoRgs4, XP_368254; MoRgs5, XP_363151; MoRgs6, XP_364773; ScSst2, NP_013557; ScRgs2, NP_014750; ScRax1, NP_014945; ScMdm1, NP_013603; MoRgs7, XP_001411659; MoRgs8, XP_001405673.

Figure 2. Comparison of various ΔMorgs mutant strains in colony morphology and conidia formation.

(A) Colony morphology was observed by incubating culture plates in the dark for ten days at 28°C and then photographed. (B) Conidia formation was observed under a light microscope 24 hours at room temperature after induction of conidiation under cover slips. (C) Comparison of specific single and double mutants in colony formation in the dark for eight days at 28°C and then photographed. (D) Comparison of specific single and double mutants in conidia formation 24 hours at room temperature after induction of conidiation under cover slips.

Figure 3. Comparison of ΔMorgs mutant strains in appressorium formation.

Appressorium formation was allowed in either inductive or non- inductive conditions. Conidia from each strain were incubated on hydrophobic (upper two panels) and hydrophilic surfaces for 24 hours (lower panel) and photographed. Black arrows indicate spores; white arrows indicate appresoria.

Figure 4. MoRgs1 and MoRgs4 are involved in sexual reproduction in M. oryzae.

Perithecia development by wild type and ΔMorgs mutant strains were photographed three weeks after inoculation. Cross between TH3 (MAT1-1) and Guy11 (MAT1-2) represents the positive control. Cross of ΔMorgs1 and ΔMorgs4 with TH3 produced either no (ΔMorgs1) or less (ΔMorgs4) peritheria and asci. ΔMorgs1/MoRGS1 and ΔMorgs4/MoRGS4 indicate complement transformants. Arrows indicate peritheria.

Figure 5. Detergent wettable phenotype of ΔMorgs1 to ΔMorgs8 mutants.

Ten microlitres of water or detergent solution containing 0.02% SDS and 5 mM EDTA were placed on the colony surfaces of the wild type and mutant strains and photographed after 5 min (Left panel). Expression analysis of MoMPG1 and MoMHP1 genes in each ΔMorgs mutant (Right panel). ΔMorgs1/MoRGS1 and ΔMorgs4/MoRGS4 indicate complement transformants. The error bars indicate SD of three replicates. Different letters in each data column indicate significant differences at P = 0.01.

Figure 6. PTH11 gene expression in ΔMorgs mutants.

RNA was extracted from mycelia cultured in liquid CM medium at 28°C for 2 days. ACTIN was used for normalization, and the values were calculated by 2^-ddCT methods with quantitative RT-PCR data. Values represent mean ± SD from two independent experiments with three replicates each.

Figure 7. MoRgs4 has a role in the regulation of extracellular laccase activities.

(A) Laccase activity was tested on CM agar medium containing 0.2 mM ABTS at final concentration. Discoloration was observed on day 2 after inoculation. (B) Laccase activity measured by ABTS oxidizing test (see Materials and Methods). (C) Quantitative RT-PCR analysis of two laccase genes in wild type and mutants. Expression data were normalized using the ACTIN gene. Error bars represent standard deviation. Different letters in each data column indicate significant differences at P = 0.01.

Figure 8. Measurement of activities of extracellular peroxidases.

(A) The discoloration of Congo red was tested on the CM agar containing 200 µg/ml of the dye. Discoloration was observed on day 7 after inoculation at 28°C. (B) Peroxidase activity measured by ABTs oxidizing test under H₂O₂ supplemented conditions. (C) Expression profiles of five extracellular peroxidase genes in the wild type and mutant strains. Different letters in each data column indicate significant differences at P = 0.01.

Figure 9. Loss of MoRGS1, MoRGS3, MoRGS4, and MoRGS7 lead to a significantly reduction in pathogenicity.

(A) Leaf spraying assay. Five milliliters of conidial suspension (5×10⁴ spores/ml) of each strain were sprayed on two-week old rice seedlings. Diseased leaves were photographed at 7 dpi. (B) Close observation of infectious growth. Excised rice sheath from 4-week-old rice seedlings was inoculated with conidial suspension (1×10⁴ spores/ml of each strain). Infectious growth was observed at 48 hpi. (C and D) ΔMorgs1ΔMorgs4 double mutant was unable to form appressorium and completely lose pathogenicity on detached barley seedling leaves. Diseased leaves were photographed 5 days after inoculation, and hyphal plugs were incubated on hydrophobic surfaces for 48 hours allowing appressorium formation.

Figure 10. MoRGS genes regulate intracellular cAMP levels during pathogenesis.

Loss of MoRGS leads to increased accumulation of total cellular cAMP levels. Bar chart showing quantification of intracellular cAMP in the mycelia of the indicated strains following 2 days of culturing in complete medium. Two biological repetitions with three replicates were assayed. The error bars represent SD of three replicates.

Figure 11. Physical interactions between RGS proteins and MoMagA, MoMagB, and MoMagC proteins.

(A) Yeast transformants expressing bait (pGBKT7) and prey (pGADT7) constructs were assayed for growth on SD-Leu-Trp-His (SD-His) plates and β-galactosidase (LacZ) activities with positive and negative control. (B) co-IP assay for the interaction of MoRgs2 with MoMagB. Western blot analysis with total proteins (Total) isolated from transformants co-expressing the MoRGS2-GFP and MoMAGB-3xFLAG constructs and proteins eluted from the anti-FLAG M2 beads (Elution). The presence of MoRgs2 and MoMagB was detected with an anti-GFP and an anti-FLAG antibody, respectively. Total proteins isolated from the wild-type strain (70-15 fractions) and detection with an anti-actin antibody was included as the controls.

Figure 12. MoRgs2 regulates asexual development upstream of MoMagB.

(A) Evaluation and quantification of conidiogenesis. Strains of the indicated genotypes were cultured in dark for 7 days at 28°C and then grown further for 3 days under constant illumination. Conidia and conidiophores were imaged under a microscope. (B) Conidiation defects in ΔMoMagB and ΔMorgs2ΔMoMagB strains. Conidia produced by the indicated strains were harvested and quantified. Data represent the mean values (±SD) from three independent experiments.