Abstract
The Rab GTPase proteins play important roles in the membrane trafficking, and consequently protein secretion and development of eukaryotic organisms. However, little is known about the function of Rab GTPases in Magnaporthe oryzae. To further explore the function of Rab GTPases, we deleted the ortholog of the yeast Sec4p protein in M. oryzae, namely MoSEC4. The ΔMosec4 mutant is defective in polarized growth and conidiation, and it displays decreased appressorium turgor pressure and attenuated pathogenicity. Notably, the biotrophic invasive hyphae produced in rice cells are more bulbous and compressed in the ΔMosec4 mutant. Further studies showed that deletion of the MoSEC4 gene resulted in decreased secretion of extracellular enzymes and mislocalization of the cytoplasmic effector PWL2-mCherry-NLS. In accordance with a role in secretion, the GFP-MoSec4 fusion protein mainly accumulates at tips of growing vegetative hyphae. Our results suggest that the MoSec4 protein plays important roles in the secretion of extracellular proteins and consequently hyphal development and pathogenicity in the rice blast fungus.
Highlights
Magnaporthe oryzae is the causal agent of rice blast disease that destroys almost a quarter of the potential global rice harvest every year (Talbot, 2003), and it is a model system for understanding microbe-plant interactions
Multiple sequence alignment indicated that all the above six proteins possess the signature motifs of Rab GTPases, including highly conserved Rabspecific residues RabF1-F5, Rab subfamily-specific sequences RabSF1-SF4, highly conserved motifs involved in guanine and phosphate/Mg2+ binding (G1-G3 and PM1-PM3), and a cysteine motif at carboxyl terminus required for subcellular localization (Figure 1)
To confirm phenotypes associated with mutation of the MoSEC4 gene, the entire ORF with its native promoter was introduced into the ∆Mosec4-11 mutant, and a complementation transformant ∆Mosec4-Com was confirmed by Southern analysis (Figures S1B,C)
Summary
Magnaporthe oryzae (synonym to Pyricularia oryzae) is the causal agent of rice blast disease that destroys almost a quarter of the potential global rice harvest every year (Talbot, 2003), and it is a model system for understanding microbe-plant interactions. M. oryzae is a hemibiotrophic fungus that penetrates the plant cuticle using a pressurized appressorium, and grows asymptomatically for about 4 days before sporulating lesions appear During this biotrophic growth phase, specialized invasive hyphae (IH) successively colonize one living rice cell after the and invaded host cells die around the time IH move into neighboring cells. For blast disease, secreted apoplastic effectors are retained inside the extrainvasive hyphal membrane (EIHM) compartment surrounding IH growing inside rice cells, resulting in outlining of the IH (Mosquera et al, 2009; Khang et al, 2010). Nonconventional secretion of cytoplasmic effectors does involve the exocyst complex, since M. oryzae mutants lacking exocyst components MoSec and MoExo show partial retention of fluorescent cytoplasmic effector protein inside BIC-associated hyphal cells, indicating inefficient secretion (Giraldo et al, 2013)
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