MoTea4-Mediated Polarized Growth Is Essential for Proper Asexual Development and Pathogenesis in Magnaporthe oryzae

Abstract

Polarized growth is essential for cellular development and function and requires coordinated organization of the cytoskeletal elements. Tea4, an important polarity determinant, regulates localized F-actin assembly and bipolar growth in fission yeast and directional mycelial growth in Aspergillus. Here, we characterize Tea4 in the rice blast fungus Magnaporthe oryzae (MoTea4). Similar to its orthologs, MoTea4-green fluorescent protein (MoTea4-GFP) showed punctate distribution confined to growth zones, particularly in the mycelial tips, aerial hyphae, conidiophores, conidia, and infection structures (appressoria) in Magnaporthe. MoTea4 was dispensable for vegetative growth in Magnaporthe. However, loss of MoTea4 led to a zigzag morphology in the aerial hyphae and a huge reduction in conidiation. The majority of the tea4⌬ conidia were two celled, as opposed to the tricellular conidia in the wild type. Structure-function analysis indicated that the SH3 and coiled-coil domains of MoTea4 are necessary for proper conidiation in Magnaporthe. The tea4⌬ conidia failed to produce proper appressoria and consequently failed to infect the host plants. The tea4⌬ conidia and germ tubes showed disorganized F-actin structures with significantly reduced numbers of cortical actin patches. Compared to the wild-type conidia, the tea4⌬ conidia showed aberrant germination, poor cytoplasmic streaming, and persistent accumulation of lipid droplets, likely due to the impaired F-actin cytoskeleton. Latrunculin A treatment of germinating wild-type conidia showed that an intact F-actin cytoskeleton is indeed essential for appressorial development in Magnaporthe. We show that MoTea4 plays an important role in organizing the F-actin cytoskeleton and is essentially required for polarized growth and morphogenesis during asexual and pathogenic development in Magnaporthe.

Fungal Morphogenesis, from the Polarized Growth of Hyphae to Complex Reproduction and Infection Structures

“…Within the growth region of the cell, the position of the polarity site may shift to change the direction of growth, but the polarity site has hitherto been viewed as a persistent feature of the growth region of the cell. This mechanism is also required the for asexual development and pathogenesis of Magnaporthe oryzae (73). The orthologous genes are conserved in other Sordariomycetes and Basidiomycota (66); however, the exact role needs to be determined.…”

A fungal monooxygenase-derived jasmonate attenuates host innate immunity

“…Mycelial plugs (wildtype strain B157 of M. oryzae obtained from the Directorate of Rice Research, Hyderabad, India) were incubated on PA in the dark at 28 °C for 3 d and then under constant illumination at room temperature for a further 5 d to induce conidiation. Conidia were harvested as described earlier 52 . Briefly, approximately 10 ml of sterile water (containing 100 mg/ml each of carbenicillin and streptomycin sulfate) was added to the conidiating colonies on PA medium, followed by gentle scraping with an inoculation loop.…”

“…The plasmid construct for Pwl2-RFP 54 Pathogenicity tests and plant-based assays. Appressorial development assay was carried out as described earlier 52 . The detached barley or rice leaf infection assays were performed to assess pathogenicity in the mutant strains.…”

Form follows function – The versatile fungal cytoskeleton

“…A number of excellent reviews have summarized the wealth of information on the various roles of F-actin and MTs during the establishment and regulation of cell polarity gathered from studies in budding and fission yeasts (Chang & Martin 2009 Landmarking proteins are either left over from previous polarization events (historical landmarks, such as Bud proteins in the budding yeast) or newly deposited to mark the site of subsequent membrane outgrowth (de novo landmarks, such as Tip Elongation Aberrant (Tea) proteins in the fission yeast and filamentous fungi) (Chang & Peter 2003;Higashitsuji et al 2009;Patkar et al 2010). Ras superfamily GTPases, including Cdc42, Rac-1 and Rho proteins, become recruited to the landmarked plasma membrane regions where their local activity is up-regulated through positive feedback mechanisms (Goryachev & Pokhilko 2008).…”

“…A well studied example of F-actin-MT cooperation, initially characterized in fission yeast but recently also demonstrated in Aspergillus nidulans, involves MT-mediated delivery of Tea proteins to the hyphal tip cortex, where they in interaction with membrane-localized proteins, such as Mod5 (Bicho et al 2010), establish stable apical clusters thought to serve as landmarks for the subsequent assembly of the F-actin cytoskeleton, and seem to increase the fidelity of polarized tip growth (Bicho et al 2010;Fischer et al 2008;Higashitsuji et al 2009;Konzack et al 2005;Sawin & Snaith 2004;Snaith & Sawin 2005;Takeshita et al 2008). POD4, a Tea1 orthologue has been identified in Neurospora crassa (Seiler & Plamann 2003), and the Tea4 orthologue of M. oryzae, MoTea4, has recently been shown to be essential for polarized growth during asexual development and pathogenicity of the rice blast fungus (Patkar et al 2010). But identification of further Tea-type orthologues in more filamentous species is still limited possibly because the protein sequence, with only w12e14 % average homology, is poorly conserved.…”