Fig. 4.

S-phase regulation during plant infection is coordinated with turgor generation. (A) Micrograph to show rice leaf sheath inoculated with Guy11, Δcds1, and Δchk1Δcds1 mutants with or without HU, added at 10 h, observed at 30 h. (Scale bar, 10 µm.) (B) Micrographs of Guy11, ∆noxR, rsy⁻, and buf⁻ mutants expressing GFP–LacI–NLS and LacO repeat construct to show effect on G1 to G2 progression. (Scale bar, 10 μm.) (C) Bar chart to show frequency of progression to G2 in appressoria of Guy11, ∆noxR, rsy⁻, and buf⁻ expressing GFP–LacI–NLS and LacO repeat construct. (D) Incipient cytorrhysis assay to measure appressorium turgor generation with or without HU. Appressoria were allowed to form on hydrophobic plastic coverslips for 10 h, when 200 mM HU was added. At 24 h, appressoria were exposed to increasing concentrations of glycerol, and the percentage of intact appressoria was recorded. *P < 0.05; **P < 0.01; ****P < 0.0001 (unpaired Student’s t test; n = 3 experiments; appressoria observed = 90–126). (E) Micrographs of Guy11 expressing Sep3–GFP, LifeAct–RFP, and Gelsolin–GFP with or without HU, added at 10 h and observed at 30 h. (Scale bar, 10 µm.) (F) Model depicting cell-cycle transitions necessary for appressorium-mediated plant infection by M. oryzae. An S-phase checkpoint operates during initial appressorium morphogenesis and depends on the DNA DDR. A second S-phase checkpoint operates during appressorium maturation and depends on cellular turgor. (G) Model to show cell-cycle control of rice cell entry by M. oryzae. A newly formed appressorium is initially arrested in G1 with a minimum turgor pressure level (Tmin). Turgor accumulates due to glycerol synthesis and melanization of the appressorium cell wall. A threshold of turgor is generated (Tmax) in the appressorium in order for S-phase entry, which is necessary for septin-dependent F-actin remodeling at the base of the appressorium. This process leads to formation of a penetration peg to rupture the plant cuticle.