Neng Zhang

The spotted alfalfa aphid (Therioaphis trifolii) is a kind of destructive pest of cultivated alfalfa (Medicago sativa) that results in significant financial losses for the livestock sector. To understand how T. trifolii navigates the biochemical defenses of its host, we investigated the effects of susceptible and resistant aphid strains on two alfalfa cultivars. T. trifolii was reared for over 50 generations on two cultivars—WL343, which is susceptible to T. trifolii, and Zhongmu No. 1, which is resistant—resulting in the development of a resistant aphid strain (R-aphid) and a susceptible aphid strain (S-aphid). The results showed that the survival rate of R-aphids was significantly higher than that of S-aphids (p = 0.039) on the resistant cultivar Zhongmu No. 1, while there was no significant difference in survival rates between the two aphid strains on WL343 (p = 0.139). This suggests that S- and R-aphids differ in their ability to modulate plant defense mechanisms, influencing their survival rates. The application of saliva from R-aphids reared on Zhongmu No. 1 (R-saliva) reduced the repellency and toxicity of treated plants, improving aphid survival. Furthermore, R-aphid infestation and R-saliva application activated the salicylic acid (SA) signaling pathway while suppressing the jasmonic acid (JA) pathway, enhancing the host plant’s suitability for aphid colonization. We propose that R-aphids may use their saliva to activate the SA pathway, which in turn inhibits JA synthesis, weakening the plant’s defenses. These findings provide valuable insights into how T. trifolii interacts with host plant defense systems.

The entomopathogen Metarhizium anisopliae is a facultative rhizosphere or endophytic fungus available for managing pests and improving plant growth. The CFEM (common in fungal extracellular membrane) proteins form a unique group in fungi but are rarely reported in entomopathogens. In this study, we cloned and identified 13 CFEM genes from M. anisopliae (MaCFEMs). Sequence alignment and WebLogo analysis showed that eight cysteines were the most conserved amino acids in their CFEM domain. Phylogenic analysis suggested that these 13 proteins could be divided into 4 clades based on the presence of the transmembrane region and the position of CFEM domain in the whole sequence. Six MaCFEM proteins with a signal peptide and without a transmembrane domain were considered candidate effector proteins. According to Phyre2 analysis, the MaCFEM88 and MaCFEM85 have the most homologous to Csa2 in Candida albicans. Subcellular localization analysis revealed that five effectors were located in the plasma membrane, while MaCFEM88 may locate in both plasma membrane and nucleus in the treated Nicotiana benthamiana. Expression pattern analysis showed that MaCFEM81, 85, 88, and 89 expression level was significantly higher in the sporulation stage compared to other growth stages. Furthermore, the yeast secretion assay showed that six candidate effectors were able to secrete out of the cell. All of the MaCFEMs couldn’t affect INF1-induced programmed cell death (PCD), but MaCFEM85 and 88 could trigger a slight hypersensitive response both when applied separately or in combination with INF1 in N. benthamiana leaves. These findings showed that six MaCFEM potential effectors with various structures and subcellular localizations in host cells might be used to illustrate the roles of MaCFEM proteins during M. anisopliae-plant interactions.