Identification and characterization of CFEM Proteins from Phakopsora pachyrhizi, the Asian soybean rust fungus

Asian soybean rust (ASR) caused by the fungus Phakopsora pachyrhizi is one of the main fungal diseases of soybean, which can cause losses of up to 95% of production under favorable environmental conditions and abundant inoculum. This pathogen is an obligate, biotrophic parasite that during the infection process secretes several effector proteins that suppress plant defense responses and promotes parasitism. One of the characteristics of fungal and oomycete effector proteins is the high content of cysteine residues (>3%). CFEM proteins (Common in Several Fungal Extracellular Membrane Proteins) are proteins secreted by different fungi species with a protein domain with 8 cysteine residues in conserved positions and diverse cellular functions. The best characterized CFEM proteins are Csa2, Rbt5, and Pga7 from the fungus Candida albicans, that are involved in a cascade to capture and absorb heme-iron from the mammalian host. The objectives of this study were to identify and characterize all CFEM proteins present in the repertoire of P. pachyrhizi fungus and to understand the biological function of these proteins as membrane component, immunity suppressor or iron capture in the pathogen life cycle. In silico analysis showed that P. pachyrhizi has 10 CFEM proteins (PpCFEM1-PpCFEM10), six of which are predicted to be secreted soluble proteins (PpCFEM1, 5, 7, 8, 9, 10) and four (PpCFEM2, 3, 4, 6) predicted as extracellular proteins with Glycosylphosphatidylinositol (GPI) docking site. Seven of the CFEM protein-coding genes (PpCFEM1-7) were expressed in the period 0 to 96 hours post-inoculation; PpCFEM2, 5, and 6 showed higher expression in the time of zero hours post-inoculation (hpi), and PpCFEM3 and 4 are expressed at 24 hpi. PpCFEM1, 3, 5, and 7 were identified in the nucleus and cytoplasm in subcellular localization assays, PpCFEM2 and PpCFEM6 proteins were identified in the cell membrane, and PpCFEM4 showed no defined subcellular localization. PpCFEM1, 4, 5, and 7 were able to suppress PTI (immunity triggered by recognition of PAPMPs), whereas only the PpCFEM4 protein was also able to suppress ETI (immunity triggered by recognition of effectors) in Nicotiana benthamiana leaves. The PpCFEM proteins did not present homology with the C. albicans CFEM proteins that participate in the heme-iron capture and absorption mechanism, and tertiary structure conformation analyses indicated that the PpCFEM proteins did not present the necessary structure for the heme-iron coordination as present in C. albicans CFEM proteins. Our findings suggest that the PpCFEM1, 4, 5, and 7 proteins can present effector molecule functions, and PpCFEM2, 3, and 6 proteins could present structural functions in the cellular process that occurs at the beginning of the infectious process. CFEM-based heme-iron capturing and absorption mechanism in P. pachyrhizi was not observed. Keywords: CFEM proteins. Phakopsora pachyrhizi. Functional characterization