Temporal Analysis of the Magnaporthe Oryzae Proteome During Conidial Germination and Cyclic AMP (cAMP)-mediated Appressorium Formation

Abstract

Rice blast disease caused by Magnaporthe oryzae is one of the most serious threats to global rice production. During the earliest stages of rice infection, M. oryzae conidia germinate on the leaf surface and form a specialized infection structure termed the appressorium. The development of the appressorium represents the first critical stage of infectious development. A total of 3200 unique proteins were identified by nanoLC-MS/MS in a temporal study of conidial germination and cAMP-induced appressorium formation in M. oryzae. Using spectral counting based label free quantification, observed changes in relative protein abundance during the developmental process revealed changes in the cell wall biosynthetic machinery, transport functions, and production of extracellular proteins in developing appressoria. One hundred and sixty-six up-regulated and 208 down-regulated proteins were identified in response to cAMP treatment. Proteomic analysis of a cAMP-dependent protein kinase A mutant that is compromised in the ability to form appressoria identified proteins whose developmental regulation is dependent on cAMP signaling. Selected reaction monitoring was used for absolute quantification of four regulated proteins to validate the global proteomics data and confirmed the germination or appressorium specific regulation of these proteins. The rice blast pathogen, Magnaporthe oryzae, is a filamentous fungus belonging to the Ascomycota. Rice blast is the most serious disease of rice worldwide and its presence has been documented in more than 85 countries including all major rice producing regions (1). Yield losses in cultivated rice associated with infection by M. oryzae can exceed 50% with typical losses ranging from 10 to 30% (1). M. oryzae also infects other grass hosts including wheat, barley and millet (2). The importance of rice as a staple food, the genetic tractability of M. oryzae, and the availability of genome sequences for both the fungus and the plant host have made the rice blast pathosystem a predominant model for the study of plantfungal interactions.Rice blast disease is initiated upon contact of an asexual conidium with the plant surface. Attachment to the leaf surface is mediated by spore tip mucilage produced at the conidial apex upon hydration of the conidium (3) followed by emergence of a germ tube from which development of a penetration structure known as an appressorium occurs in response to plant-derived cues. Appressoria of M. oryzae are characterized by a heavily melanized cell wall that facilitates, via accumulation of compatible solutes, generation of a large internal turgor pressure that provides the mechanical force required for penetration of the plant cuticle by a penetration peg formed at the appressorium pore (4). Proper development of the appressorium is essential for infection and understanding the biology of appressorium formation is critical for the development of control strategies for the rice blast disease.Appressorium formation can be induced in vitro by germination of conidia on...

Phosphoproteome Analysis Links Protein Phosphorylation to Cellular Remodeling and Metabolic Adaptation during Magnaporthe oryzae Appressorium Development

“…A total of 1514 unique phosphoproteins were identified from 4894 phosphorylation sites including a subset of 2924 class I phosphorylation sites where the localization of the phosphorylation site was confidently assigned as defined previously [56,57]. The complete list of phosphosite identifications from the Phospho(STY) output file generated by MaxQuant is included here as Supplementary Table 1.…”

“…M. oryzae wild type strain 70-15 and a ΔcpkA mutant 57 were routinely maintained on minimal medium agar 57 at 25°C. Mycelial samples were harvested from 50 mL minimal medium broth cultures grown in 250 mL shake flasks for five days at 25°C and flash frozen in liquid nitrogen.…”

“…Additional aliquots of 1.8 million conidia were germinated on the hydrophilic surface of 205 × 110 mm GelBond ® (Lonza, Rockland, ME) sheets in 16 mL of H 2 O. Following eight hours of germination, germinated conidia were harvested from three sheets of GelBond ® as described previously 57 . Additional eight hour germinated samples were treated with 10 µM 1,16-hexadecanediol (wild type and ΔcpkA mutant strains) or mock treated with ethanol (wild type only) via the addition of 4 mL of H 2 O spiked with 20 µL of a 10 mM 1,16-hexadecanediol solution (in 100% ethanol) or 20 µL of 100% ethanol.…”

Characterization of 47 Cys2‐His2 zinc finger proteins required for the development and pathogenicity of the rice blast fungus Magnaporthe oryzae

“…(), were also significantly down‐regulated in the Δ vrf1 appressoria (Table S11). The gene expression profile of the Δ vrf1 appressoria and GO enrichment analysis showed a significant change in expression of genes involved in cell wall construction, carbohydrate metabolism, lipid metabolism, protein degradation, cellular transportation and response to oxidative stress; and the genes related to these biological processes were previously found to be significantly altered during appressorium formation (Oh et al ., ; Soanes et al ., ; Franck et al ., ). Although the expression of 2701 genes was affected by Vrf1 in appressoria, most of them were not affected in mycelia (Fig.…”

Subcellular compartmentation, interdependency and dynamics of the cyclic AMP‐dependent PKA subunits during pathogenic differentiation in rice blast

“…Inspite of such overall conservation, each fungal system has its own signalling specificities and controls different functions. Magnaporthe oryzae is an economically important ascomycetous fungus that causes the devastating rice blast disease with typical yield losses ranging from 10 to 30% (Franck et al, 2013). M. oryzae is also able to infect other cereals like wheat, barley and millet and the rice-blast pathosystem serves as an excellent model to investigate the fungus-host interactions (Valent, 1990).…”