Common Genetic Pathways Regulate Organ-Specific Infection-Related Development in the Rice Blast Fungus

Abstract

Magnaporthe oryzae is the most important fungal pathogen of rice (Oryza sativa). Under laboratory conditions, it is able to colonize both aerial and underground plant organs using different mechanisms. Here, we characterize an infection-related development in M. oryzae produced on hydrophilic polystyrene (PHIL-PS) and on roots. We show that fungal spores develop preinvasive hyphae (pre-IH) from hyphopodia (root penetration structures) or germ tubes and that pre-IH also enter root cells. Changes in fungal cell wall structure accompanying pre-IH are seen on both artificial and root surfaces. Using characterized mutants, we show that the PMK1 (for pathogenicity mitogen-activated protein kinase 1) pathway is required for pre-IH development. Twenty mutants with altered pre-IH differentiation on PHIL-PS identified from an insertional library of 2885 M. oryzae T-DNA transformants were found to be defective in pathogenicity. The phenotypic analysis of these mutants revealed that appressorium, hyphopodium, and pre-IH formation are genetically linked fungal developmental processes. We further characterized one of these mutants, M1373, which lacked the M. oryzae ortholog of exportin-5/Msn5p (EXP5). Mutants lacking EXP5 were much less virulent on roots, suggesting an important involvement of proteins and/or RNAs transported by EXP5 during M. oryzae root infection.

Tissue-Adapted Invasion Strategies of the Rice Blast Fungus Magnaporthe oryzae

“…Fungal hyphae entered the tissue mainly through rhizodermal cells, but rare penetration of root hairs was also observed ( Figure 1C). Consistent with previous reports, the melanized appressoria that are typically associated with leaf infection were not observed on roots (Sesma and Osbourn, 2004;Tucker et al, 2010). Instead, penetration pegs were formed from swollen hyphal structures reminiscent of hyphopodia ( Figure 1B).…”

“…For example G. graminis, the economically important take-all pathogen of wheat, is a well-studied relative of M. oryzae that invades roots via hyphopodia that form on hyphae colonizing the root surface (Asher and Shipton, 1981). As could have been expected, M. oryzae also initiates root infection from simple hyphopodia without formation of a specialized infection structure (Sesma and Osbourn, 2004;Tucker et al, 2010). However, M. oryzae employs an additional developmental program for appressoria differentiation during leaf infection, a feature that G. graminis, for example, lacks.…”

“…The ability to use either hyphopodia or appressoria thus permits M. oryzae to adapt its penetration mechanisms to the properties of the target organ, which in consequence raises questions as to the degree of similarity between leaf and root infection strategies. Over recent years it has become clear that the fungal genetic tool box of M. oryzae includes a combination of common and organ-specific components to accomplish initial penetration of either the cuticlecovered leaf epidermis or the rhizodermis (Dufresne and Osbourn, 2001;Sesma and Osbourn, 2004;Tucker et al, 2010). However, the intraradical development of the fungus has not been fully characterized.…”

“…However, the intraradical development of the fungus has not been fully characterized. Standard microscopy investigations have lead to the suggestion that the fungus passes the rhizodermis and the root cortex by inter-and intracellular growth, likely following a hemibiotrophic strategy comparable to that in leaves (Sesma and Osbourn, 2004;Heupel et al, 2010;Tucker et al, 2010). Furthermore, the fungus has been observed finally to enter into the central cylinder of the root from where it systemically spreads throughout the plant (Sesma and Osbourn, 2004), and, following prolonged cocultivation of 2 to 4 weeks, blast symptoms have been observed on both roots and on aerial organs of root-inoculated plants, thereby revealing that although symptom development occurs considerably slower than on leaves, the fungus has the ability to cause disease on roots and systemically (Sesma and Osbourn, 2004).…”

Fusion of a Novel Genetically Engineered Chitosan Affinity Protein and Green Fluorescent Protein for Specific Detection of Chitosan In Vitro and In Situ

“…One possibility for the specific detection of polysaccharides are polysaccharide hydrolases or other polysaccharide-modifying enzymes with an affinity to the targeted molecule, or specific polysaccharide binding proteins or modules (16,19,28,32,35), an approach already successfully employed for the detection of chitosan (17). Such proteins can either be detected by using specific antibodies directed against them (12,33,38) or by tagging the proteins chemically, e.g., using fluorescence tags such as fluorescein isothiocyanate (FITC) or using gold particles (4-6, 17, 26), or genetically, by generating fusion proteins, e.g., with the green fluorescent protein (GFP) (19) or with a peptide tag against which commercial antibodies are available. We here describe an extension of the latter method, generating by genetic engineering a chitosan affinity protein (CAP) based on a bacterial chitosanase (CSN) that we inactivated using sitedirected mutagenesis and to which we fused three different tags, namely, enhanced GFP (eGFP) and the affinity tags StrepII and His 6 , for purification and detection.…”