Its expression peaked during haustorium formation, which is similar to the expression pattern of PtMAPK1 in P. triticina during plant infection. These observations suggest that the development of highly specialized infection structures such as haustoria in rust fungi is regulated by a well conserved MAPK signaling cascade. Expression of the PsMAPK1 gene also partially restored the defects of the F. graminearum map1 mutant in vegetative growth and plant infection. The fact that the YERK1 subfamily genes are highly conserved may explain for observed functional relatedness among pathogens with different plant infection mechanisms, such as F. graminearum, M. oryzae, and Pst. However, the phenotypes of the map1 and pmk1 mutants were only partially complemented, indicating that PsMAPK1 is not fully functional in ascomycetous fungi. Pst is a rust pathogen that has a distinct life style from M. oryzae and F. graminearum. During evolution, sequence and structural changes in PsMAPK1 may enable it to interact with other components of this MAPK pathway that are not conserved. These changes may reduce the efficiency of PsMAPK1 in signal transduction in ascomycetes and account for partial complementation. Complementation assays with the MAPK mutants of the basidiomycetous pathogen U. maydis may be better for functional analysis with PsMAPK1. However, the PtMAPK1 gene from P. triticina also only partially complemented the U. maydis kpp2 mutant. Sequence alignment revealed that PsMAPK1 shares 77%, 74%, 75%, and 75% amino acid sequence identity with Kpp6 and Kpp2 of U. maydis, Pmk1 of M. oryzae, and Map1 of F. graminearum, respectively. Therefore, the overall homology of PsMAPK1 with its orthologs from U. maydis is not significantly higher than with its orthologs from two ascomycetes. Although PsMAPK1 partially rescued the pmk1 mutant for appressorium formation, no GFP signals could be detected in appressoria formed by transformant CM-10. A similar observation has been reported by Yang and colleagues. Although expression of a COM1-eGFP fusion construct complemented the com1 deletion mutant, GFP signals were not detectable in vegetative hyphae, conidia, germination tubes, appressoria, or infection hyphae of M. oryzae. The abundance of the PsMAPK1- eGFP fusion proteins may be too low to be detected by fluorescence microscopy in these transformants. However, it is more likely that the PsMAPK1-eGFP fusion proteins are not stable or lack fluorescent signals. Fusion with the PsMAPK1 protein may change the structure of GFP proteins. In qRT-PCR assays, PsMAPK1 was highly Reversine expressed during the haustorium formation stage. However, its expression was not significantly up-regulated from 6 to 12 hpi, which corresponded to the appressorium formation stage. There are contradictory reports on the formation of appressoria by Pst in penetration of wheat stomata.