Bacillus subtilis KLBMPGC81 suppresses appressorium-mediated plant infection by altering the cell wall integrity signaling pathway and multiple cell biological processes in Magnaporthe oryzae

Abstract

Magnaporthe oryzae is one of the most destructive crop pathogens in the world, causing huge losses in rice harvest every year. Bacillus subtilis is a potential biocontrol agent that has been explored in many crop systems because it is a potent producer of bioactive compounds. However, the mechanisms by which these agents control rice blasts are not fully understood. We show that B. subtilis KLBMPGC81 (KC81) and its supernatant (SUP) have high antimicrobial activity against M. oryzae strain Guy11. To better exploit KC81 as a biocontrol agent, the mechanism by which KC81 suppresses rice blast pathogens was investigated. This study shows that KC81 SUP is effective in controlling rice blast disease. The SUP has a significant effect on suppressing the growth of M. oryzae and appressorium-mediated plant infection. KC81 SUP compromises cell wall integrity, microtubules and actin cytoskeleton, mitosis, and autophagy, all of which are required for M. oryzae growth, appressorium development, and host infection. We further show that the SUP reduces the activity of the cyclin-dependent kinase Cdc2 by enhancing the phosphorylation of Cdc2 Tyr 15, thereby impairing mitosis in M. oryzae cells. SUP induces the cell wall sensor MoWsc1 to activate the cell wall integrity pathway and Mps1 and Pmk1 mitogen-activated protein kinases. Taken together, our findings reveal that KC81 is an effective fungicide that suppresses M. oryzae growth, appressorium formation, and host infection by abnormally activating the cell wall integrity pathway, disrupting the cytoskeleton, mitosis, and autophagy.

Keywords: appressorium; autophagy; bacillus subtilis; biocontrol; cell cycle; cell wall integrity; magnaporthe oryzae; plant infection.

Figure 1

Biocontrol of M. oryzae using B subtilis KC81. (A) Cultured strains Guy11 on CM plates with B subtilis KC81. (B) Cultured strains Guy11 on CM plates with KC81 SUP. (C) Leaves showing blast symptoms in the presence of KC81 SUP. All rice seedlings were treated with strain Guy11. Guy11: M. oryzae; SUP: cell-free culture supernatants of strain KC81; SUP (1 day later) and SUP (1 day before): SUP sprayed onto rice seedlings 1 day after or 1 day before the strain Guy11 spray treatments, respectively; SUPM: rice seedlings sprayed with strain Guy11 mixed with SUP.

Figure 2

KC81 SUP impairs appressorium development and function of M. oryzae. (A) Appressorium formation assay on hydrophobic surfaces. Conidia were incubated on hydrophobic surfaces and the samples were observed. (B) Statistical analysis of spore germination rates and appressorium formation rates at different time points. The percentage at a given time was recorded by observing at least 100 conidia for each strain and the experiment was repeated thrice. Error bars represent SD and two asterisks represent significant differences (p < 0.01). (C) Appressorium formation assay on rice leaves. Conidia were incubated on rice leaves and the samples were observed after 24 (h) (D) Microscopic observation of infectious growth on barley. Excised barley leaves from 7-day-old barley seedlings were inoculated with conidial suspension (5 × 10⁴ spores/mL). Infectious growth was observed at 24 h and 48 h post-inoculation. Bar = 20 μm.

Figure 3

KC81 SUP destroys cell wall integrity of M. oryzae. KC81 SUP altered the distribution of chitin in the cell wall of M. oryzae. Hyphae were stained with 10 μg/mL calcofluor white (CFW) for 5 min without light before being photographed. Bar = 50 μm.

Figure 4

KC81 SUP impairs actin cytoskeleton organization of M. oryzae. (A) Live cell imaging experiment to show MoAbp1-GFP (green fluorescent protein) localization in M. oryzae mycelium in the presence or absence of KC81 SUP. (B) Live cell imaging experiment to show MoAbp1-GFP localization during M. oryzae appressorium development in the presence or absence of KC81 SUP. Bar = 10 μm.

Figure 5

KC81 SUP impairs microtubule cytoskeleton organization of M. oryzae. (A) Live cell imaging experiment to show MoTub2-GFP localization in M. oryzae mycelium in the presence or absence of KC81 SUP. (B) Live cell imaging experiment to show MoTub2-GFP localization during M. oryzae appressorium development in the presence or absence of KC81 SUP. Bar = 10 μm.

Figure 6

KC81 SUP impairs mitosis of M. oryzae. (A) Live cell imaging experiment to show MoH1-RFP (red fluorescent protein) localization in M. oryzae mycelium in the presence or absence of KC81 SUP. (B) Live cell imaging experiment to show MoH1-RFP localization during M. oryzae appressorium development in the presence or absence of KC81 SUP. Bar = 10 μm.

Figure 7

KC81 SUP promotes cell autophagy of M. oryzae. (A) Live cell imaging experiment to show GFP-Atg8 localization in M. oryzae mycelium in the presence or absence of KC81 SUP. (B) Immunoblotting was performed with anti-GFP antibodies. The extent of autophagy was estimated by calculating the amount of free GFP compared with the total amount of intact GFP-Atg8 and free GFP. Bar = 10 μm.

Figure 8

Cell wall integrity signaling pathway response proteins MoWsc1 respond to KC81 SUP. (A) Live cell imaging experiment to show MoWsc1-GFP localization in M. oryzae mycelium in the presence or absence of KC81 SUP. (B) Live cell imaging experiment to show MoWsc1-GFP localization during M. oryzae appressorium development in the presence or absence of KC81 SUP. Bar = 10 μm.

Figure 9

KC81 SUP activates phosphorylation of Mps1-, Pmk1- MAPK, and MoCdc2. (A) Western blot assays for phosphorylation of Mps1 and Pmk1. (B) Western blot assays for phosphorylation of Cdc2.