Secreted Glycosyltransferase RsIA_GT of Rhizoctonia solani AG-1 IA Inhibits Defense Responses in Nicotiana benthamiana

Abstract

Anastomosis group AG-1 IA of Rhizoctonia solani Khün has a wide host range and threatens crop production. Various glycosyltransferases secreted by phytopathogenic fungi play an essential role in pathogenicity. Previously, we identified a glycosyltransferase RsIA_GT (AG11A_09161) as a secreted protein-encoding gene of R. solani AG-1 IA, whose expression levels increased during infection in rice. In this study, we further characterized the virulence function of RsIA_GT. It is conserved not only in Basidiomycota, including multiple anastomosis groups of R. solani, but also in other primary fungal taxonomic categories. RsIA_GT possesses a signal peptide (SP) for protein secretion, and its functionality was proven using yeast and Nicotiana benthamiana. The SP-truncated form of RsIA_GT (RsIA_GT(ΔS)) expressed in Escherichia coli-induced lesion-like phenotype in rice leaves when applied to punched leaves. However, Agrobacterium-mediated transient expressions of both the full-length RsIA_GT and RsIA_GT(ΔS) did not induce cell death in N. benthamiana leaves. Instead, only RsIA_GT(ΔS) suppressed the cell death induced by two reference cell death factors BAX and INF1 in N.benthamiana. RsIA_GT(ΔS)^{R154A D168A D170A}, a mutant RsIA_GT(ΔS) for the glycosyltransferase catalytic domain, still suppressed the BAX- or INF1-induced cell death, suggesting that the cell death suppression activity of RsIA_GT(ΔS) would be independent from its enzymatic activity. RsIA_GT(ΔS) also suppressed the H₂O₂ production and callose deposition and showed an effect on the induction of defense genes associated with the expression of BAX and INF1. The transient expression of RsIA_GT(ΔS) in N. benthamiana enhanced the lesion area caused by R. solani AG-1 IA. The secreted glycosyltransferase, RsIA_GT, of R. solani AG-1 IA is likely to have a dual role in virulence inside and outside of host cells.

Keywords: glycosyltransferases; pathogenicity; phytopathogenic fungi; plant innate immunity; virulence.

Figure 1

The phylogenetic tree of RsIA_GT and glycosyltransferase in other basidiomycetes. The tree with the highest log likelihood (−7084.07) is shown. Initial tree(s) for the heuristic search was obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Whelan and Goldman + Freq. model and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Numerals on branches indicate probabilities with 1000 bootstrap trials. A red asterisk indicates RsIA_GT.

Figure 2

Prediction and functional validation of the signal peptide (SP) of RsIA_GT: (a) The score provided by SignalP 3.0 for the SP analysis of RsIA_GT. The 27 amino acid residues of the N-terminus of RsIA_GT were predicted as an SP. (b) Functional validation of SP of RsIA_GT by yeast secretion system (YST). (top) Yeast strains containing pSUC2 vectors with or without functional SP could grow on the CMD-W medium containing sucrose and glucose as carbon sources but not tryptophan for vector selection. The SPs of Mg87 protein of M. oryzae and Avr1b protein of P. sojae are the negative and the positive control, respectively. (middle) Only yeast containing pSUC2 vectors with functional SP could grow on the YPRAA medium. (bottom) Validation of invertase activity outside the yeast cells with 2, 3, 5-triphenyltetrazlium chloride (TTC). The supernatants of the liquid culture medium of each yeast strain were supplemented with colorless TCC. The invertase activity can be detected as the red color of 1,3,5-triphenylformazan (TPF) by the conversion of TTC to TPF with invertase enzymatic activity. (c,d) Functional validation of the SP of the RsIA_GT using INF1-induced cell death in N. benthamiana leaves as an indicator by transient expression of Agrobacterium syringe infiltration method. The photograph was taken at four days after the infiltration (c), and it was subsequently stained with trypan blue (d). RsIA_GT without SP and green fluorescent protein (GFP) served as the negative control. INF1 served as the positive control. (e) Western blot confirmation expression of INF1, SP(RsIA_GT)-C-INF1, RsIA_GT, and GFP using anti-FLAG antibody.

Figure 3

RsIA_GT(ΔS) induces chlorosis after the treatment with wounded rice leaves: (a) SDS-PAGE of the soluble fraction of protein extracts of the uninduced (lanes 1 and 2) and the induced (lane 3) E. coli BL21 strain expressing RsIA_GT(ΔS)-MBP. A red asterisk shows the expected size of the target protein (77.74 kDa). (b) Rice leaves treated with the extracted proteins (20 μg/mL, not purified) of E. coli expressing plasmids harboring RsIA_GT(ΔS)-MBP or MBP (CK: the negative control). The photographs were taken at five days after the treatments. (c) Lesion-like area in rice leaves induced by treating the protein extract of E. coli BL21 expressing RsIA_GT(ΔS)-MBP at five days after the treatment. The areas were quantified using ImageJ software. Data represent mean ± SE of three independent biological replicates (** p < 0.01, Student’s t-test).

Figure 4

Evaluation of the cell death-inducing activity and the immunity-suppressing activity of RsIA_GT and RsIA_GT(ΔS) in Nicotiana benthamiana using Agrobacterium-mediated transient expression analysis: (a,b) Visible necrotic phenotype (a) and trypan-blue stained cell death (b) induced by the transient expressions of RsIA_GT, RsIA_GT(ΔS), BAX, and INF1. GFP served as the negative control. The photograph was taken at four days after the infiltration. Both RsIA_GT and RsIA_GT(ΔS) did not induce cell death in N. benthamiana. (c) Western blot confirmation of expression of RsIA_GT, RsIA_GT(ΔS), BAX, INF1, and GFP using anti-FLAG antibody. (d,e) Visible necrotic phenotype (d) and trypan-blue stained cell death (e) after the co-expression of RsIA_GT and RsIA_GT(ΔS) with BAX or INF1. GFP served as the negative control. The photograph was taken at four days after the infiltration. (f) Western blot confirmation of co-expression of RsIA_GT, RsIA_GT(ΔS), and GFP with BAX or INF1 using anti-FLAG antibody. The blue asterisks indicate the protein bands of BAX. The green asterisks indicate the protein bands of INF1. The red asterisks indicate the protein bands of the correct size. Both RsIA_GT and RsIA_GT(ΔS) did not induce cell death in N. benthamiana. Only RsIA_GT(ΔS) suppressed the HR cell death induced by BAX and INF1 in N. benthamiana.

Figure 5

RsIA_GT(ΔS) suppresses the product of H₂O₂ and callose as well as the expression of the defense-related genes induced by BAX and INF1 in N. benthamiana: (a–d) Detection of H₂O₂ by DAB staining method (a,b) and callose by aniline blue staining method (c,d) in the leaves agroinfiltrated with BAX (left), BAX and GFP (middle), and BAX and RsIA_GT(ΔS) (right). GFP served as a negative control. The leaves were sampled at 12 h (a,b) and 24 h (c,d) after the agroinfiltrations and used for the staining experiments, respectively. Bar (a and b), 2 mm. Bar (c,d), 10 μm. The white and red arrows indicate the representative areas for the accumulations of H₂O₂ and callose, respectively. (e,f) Expression analysis of defense-related genes, RbohB, PR4a, and WRKY12 in N. benthamiana leaves agroinfiltrated with BAX and RsIA_GT(ΔS) (e) and INF1 and RsIA_GT(ΔS) (f). GFP was used as a negative control. The leaves were collected at 12 h and 36 h after the agroinfiltrations and used for RNA extraction to synthesize cDNA for the qRT-PCR analysis. The experiments were repeated three times with independent biological samples. ** p < 0.01, Student’s t-test.

Figure 6

Conservation of key amino acid residues of glycosyltransferase catalytic domain in RsIA_GT and assessment of their requirements for the suppression activity of HR cell death: (a) Alignment of the amino acid sequences of glycosyltransferase catalytic domain of RsIA_GT and eight large clostridial toxins: LT (X82638 from Clostridium sordellii strain 6018), Toxin_B (X53138 from Clostridioides difficile strain VPI 10463), Toxin_BF (Z23277 from C. difficile strain 1470), Toxin_A (M30307 from C. difficile strain VPI 10463), α-Toxin (Z48636 from Clostridium novyi), Ochlp (D11095 from Saccharomyces cerevisiae), Surlp (M96648 from S. cerevisiae), and Mnnlp (L23753 from Saccharomyces cerevisiae). The red asterisks indicate the key amino acid residues of glycosyltransferase catalytic domain. (b,c) Co-expression analysis of RsIA_GT(ΔS), RsIA_GT(ΔS)^{R154A D168A D170A}, and GFP with INF1 or BAX in N. benthamiana leaves for the evaluation of the requirement of the key amino acids for the immune suppression activity of RsIA_GT(ΔS). The indicated combinations of genes were agroinfiltrated, and necrosis (b) and cell death detected with trypan blue staining (c) were observed at three days after the treatment. GFP served as a negative control. (d) Western blot confirming the co-expression of RsIA_GT(ΔS), RsIA_GT(ΔS)^{R154A D168A D170A}, and GFP with BAX or INF1 using an anti-FLAG antibody. The blue asterisks indicate the protein bands of BAX. The green asterisks indicate the protein bands of INF1. The red asterisks indicate the protein bands of the correct size.

Figure 7

Transiently expressed RsIA_GT(ΔS) enhanced the symptom formation caused by R. solani AG-1 IA in N. benthamiana: (a,b) R. solani AG-1 IA was inoculated to the leaves of N. benthamiana at 2 days after the filtration with Agrobacterium harboring RsIA_GT(ΔS) or GFP. The lesion formation was observed at three days after the inoculation. (c) The lesion area was quantified using the ImageJ software. Data were mean ± SE of three independent biological replicates (** p < 0.01, Student’s t-test).