Using transmission electron microscopy analysis of rice sheath cells infected with a pathogenic M. oryzae strain, the authors confirmed the previously observed constriction of invasive hyphae as they cross the plant cell wall. Moreover, callose deposition (a plant defence mechanism that leads to closure of intercellular plasmodesmata) was not observed at early stages of infection (that is, 27 hours post-inoculation), which suggests that M. oryzae inhibits the deposition of plasmodesmal occlusion materials. To investigate the molecular mechanisms that control invasive growth, the authors used a chemical genetic approach to inactivate the fungal MAPK Pmk1, which was previously shown to be crucial for appressorium development and pathogenicity. Following invasion of the first rice epidermal cell, the authors inhibited Pmk1, which prevented the invasion of adjacent epidermal cells, as indicated by the observation that the first infected cell became filled with fungal hyphae. This suggests that Pmk1 is crucial for cell-to-cell spread and invasive growth. RNA sequencing (RNA-seq) analysis revealed that the expression of 1,457 fungal genes was altered in cells in which Pmk1 was inhibited compared with cells in which Pmk1 activity was not suppressed. A subset of genes that were positively regulated by Pmk1 encode fungal effectors implicated in the suppression of plant immunity, including several Bas effectors, which might function at cell wall crossing sites. Indeed, Bas2–GFP and Bas3–GFP were not expressed in fungal cells in which Pmk1 was inhibited. Moreover, Pmk1 inhibition was accompanied by increased reactive oxygen species-dependent callose deposition.
The RNA-seq analysis also revealed that morphogenetic regulators are downregulated in the fungus during Pmk1 inhibition, including factors that regulate the dynamics of septins and filamentous (F)-actin, which form a ring-shaped structure at the base of the appressorium to promote the penetration of the rice leaf. Septin mutants did not penetrate the rice leaf efficiently, and in cases in which penetration was successful, the mutants had a decreased ability to spread between rice cells, which is consistent with a role for septins in cell-to-cell invasion by regulating the constriction of invasive hyphae of M. oryzae.
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