Phylogeny of Regulators of G-Protein Signaling Genes in Leptographium qinlingensis and Expression Levels of Three RGSs in Response to Different Terpenoids

Abstract

Leptographium qinlingensis is a bark beetle-vectored pine pathogen in the Chinese white pine beetle (Dendroctonus armandi) epidemic in Northwest China. L. qinlingensis colonizes pines despite the trees' massive oleoresin terpenoid defenses. Regulators of G-protein signaling (RGS) proteins modulate heterotrimeric G-protein signaling negatively and play multiple roles in the growth, asexual development, and pathogenicity of fungi. In this study, we have identified three L. qinlingensis RGS genes, and the phylogenetic analysis shows the highest homology with the regulators of G-protein signaling proteins sequence from Ophiostoma piceae and Grosmannia clavigera. The expression profiles of three RGSs in the mycelium of L. qinlingensis treated with six different terpenoids were detected, as well as their growth rates. Under six terpenoid treatments, the growth and reproduction in L. qinlingensis were significantly inhibited, and the growth inflection day was delayed from 8 days to 12-13 days. By analyzing the expression level of three RGS genes of L. qinlingensis with different treatments, results indicate that LqFlbA plays a crucial role in controlling fungal growth, and both LqRax1 and LqRgsA are involved in overcoming the host chemical resistances and successful colonization.

Keywords: Leptographium qinlingensis; expression; host resistances; regulators of G-protein signaling; terpene tolerance.

Figure 1

Phylogenetic tree of three regulators of G-protein signaling (RGS) from L. qinlingensis. The sequences from L. qinlingensis are marked in black. The phylogenetic tree was constructed with MEGA-X using the neighbor-joining method. Values indicated at the nodes are bootstrap values based on 1000 replicates.

Figure 2

Structure of RGSs in L. qinlingensis. (A) Alignment of LqFlbA sequences and the consensus sequences in other species, including: O. piceae UAMH 11346 (OpFlbA, EPE08676.1), S. brasiliensis 5100 (SbFlbA, XP_040618117.1), S. schenckii ATCC 58251 (SsFlbA-1, ERS97713.1), S. schenckii 1099-18 (SsFlbA-2, XP_016584922.1), S. insectorum RCEF 264 (SiFlbA, OAA58369.1), P. minimum UCRPA7 (PmFlbA, XP_007911287.1), and G. clavigera kw1407 (GcFlbA, XP_014168425.1), both of which contain two copies of the DEP domain and a C-terminal RGS domain; (B) Alignment of LqRax1 sequences and the consensus sequences in other species, including: O. piceae UAMH 11346 (OpRax1, EPE03546.1), S. insectorum RCEF 264 (SiRax1, OAA68205.1), S. schenckii ATCC 58251 (SsRax1-1, ERS99229.1), S. schenckii 1099-18 (SsRax1-2, XP_016585765.1), S. brasiliensis 5100 (SbRax1, XP_040622306.1), G. clavigera kw1407 (GcRax1, XP_014168658.1), and C. sp. RAO-2017 (CsRax1, PHH84815.1), both of which contain an N-terminal RGS domain and three DEP domains transmembrane domains; (C) Alignment of LqRgsA sequences and the consensus sequences in other species, including: O. piceae UAMH 11346 (OpRgsA, EPE03546.1), S. insectorum RCEF 264 (SiRgsA, OAA68205.1), S. schenckii ATCC 58251 (SsRgsA-1, ERS99229.1), S. schenckii 1099-18 (SsRgsA-2, XP_016585765.1), S. brasiliensis 5100 (SbRgsA, XP_040622306.1), G. clavigera kw1407 (GcRgsA, XP_014168658.1). and C. sp. PMI_546 (CsRgsA, KAH8906889.1), both of which contain an N-terminal RGS domain and three DEP domains transmembrane domains and are similar to that of Rax1. Identical amino acid residues in all proteins are shown in black; pink parts indicate more than 75% identical amino acids, and blue parts indicate more than 50% identical amino acids. Rad frames: RGS domains; Blue frames: DEP domains; Green frames: transmembrane region.

Figure 3

Growth rate of L. qinlingensis in different terpenoid treatments. The growth condition was obtained by calculating the area of the colony. The results represent the mean ± SE of five independent experiments. (A) 5% concentration treatments; (B) 10% concentration treatments; (C) 20% concentration treatments.

Figure 4

Quantitative expression of the three RGS genes (mean ± SE) in L. qinlingensis following treatment with different terpenoids. (A) Relative expression of LqFlbA; (B) Relative expression of LqRax1; (C) Relative expression of LqRgsA. RGSs expressions were normalized with EF1 gene. The 2^−ΔΔCt and SE values were transformed at log₂ for plotting. Different letters indicate significant differences at p < 0.05 (Tukey test, no letter means no significant difference) among different concentrations of the same stimulus. Mycelial growth on MEA medium with 200 mL DMSO was set as the control group (CK). Based on the value of CK (X-axis), the expression levels of other treatments are higher than CK as a positive value (expression up-regulation), and vice versa for a negative value (expression down-regulation).