Bypassing Both Surface Attachment and Surface Recognition Requirements for Appressorium Formation by Overactive Ras Signaling in Magnaporthe oryzae

Abstract

Magnaporthe oryzae forms a highly specialized infection structure called an appressorium for plant penetration. In M. oryzae and many other plant-pathogenic fungi, surface attachment and surface recognition are two essential requirements for appressorium formation. Development of appressoria in the air has not been reported. In this study, we found that expression of a dominant active MoRAS2(G18V) allele in M. oryzae resulted in the formation of morphologically abnormal appressoria on nonconducive surfaces, in liquid suspensions, and on aerial hyphae without attachment to hard surfaces. Both the Pmk1 mitogen-activated protein kinase cascade and cAMP signaling pathways that regulate surface recognition and appressorium morphogenesis in M. oryzae were overactivated in the MoRAS2(G18V) transformant. In mutants deleted of PMK1 or CPKA, expression of MoRAS2(G18V) had no significant effects on appressorium morphogenesis. Furthermore, expression of dominant MoRAS2 in Colletotrichum graminicola and C. gloeosporioides also caused the formation of appressorium-like structures in aerial hyphae. Overall, our data indicate that MoRas2 functions upstream from both the cAMP-PKA and Pmk1 pathways and overactive Ras signaling leads to improper activation of these two pathways and appressorium formation without surface attachment in appressorium-forming pathogens.

The cAMP-PKA Signaling Pathway Regulates Pathogenicity, Hyphal Growth, Appressorial Formation, Conidiation, and Stress Tolerance in Colletotrichum higginsianum

“…In M. oryzae , the feedback between the cAMP-PKA and MAPK signaling pathways regulates appressorium morphogenesis and plant infection (Zhou et al, 2012, 2014). In C. lagenarium , cAMP-PKA signaling pathway cooperated with MAPK to regulate appressorial formation and infectious growth (Takano et al, 2000; Yamauchi et al, 2004).…”

The farnesyltransferase β‐subunit RAM1 regulates localization of RAS proteins and appressorium‐mediated infection in Magnaporthe oryzae

“…Ras proteins are small GTPases which can respond to external stimuli and activate various downstream signalling pathways for cellular responses (Zhou et al , ) and whose subcellular localization is important for their functions (Prior and Hancock, ). Interestingly, Ras proteins were widely reported to be the farnesylation targets in eukaryotic cells, so we sought to determine if the functions of M. oryzae Ras proteins are regulated by farnesylation.…”

“…Some Ras‐like proteins were found in M. oryzae , and they were used to perform farnesylation site prediction by GPS‐Lipid (http://lipid.biocuckoo.org/webserver.php) (Xie et al , ). Six RAS‐like proteins, Ras1, Ras2, rho1, rho2, rho3, rho4 and ced‐10 (Fu et al , ; Zheng et al , ; Zhou et al , ), were found to contain the C‐terminal CaaX motifs (Fig. , see Supporting Information), suggesting that they could be regulated by farnesylation.…”

“…, see Supporting Information), suggesting that they could be regulated by farnesylation. We chose Ras1 and Ras2, which have been reported to be putative regulatory factors of cAMP and PMK1‐MAPK signalling pathways in different fungi (Tamanoi, ; Zhou et al , ), for validation.…”

“…In S. cerevisiae , two RAS proteins, Ras1 and Ras2, both control the cAMP‐PKA signalling pathway (Tamanoi, ). RAS2 is also reported to function upstream of both of the cAMP and Pmk1‐MAPK signalling pathways in M. oryzae to regulate appressorium formation (Zhou et al , ). Normally, the location of the Ras proteins on the plasma membrane is essential for their functions.…”