Glycoside Hydrolase Family 16 Enzyme RsEG146 From Rhizoctonia solani AG1 IA Induces Cell Death and Triggers Defence Response in Nicotiana tabacum

Abstract

Rhizoctonia solani AG1 IA is a harmful necrotrophic fungus responsible for various crop diseases, including maize and rice sheath blight, which can lead to significant production losses. However, the pathogenic mechanisms and the roles of effectors in this pathogen remain poorly understood. In this study, we identified a glycoside hydrolase 16 family gene, RsEG146, from R. solani that was upregulated during its infection of Zea mays leaves. When transiently expressed through agroinfiltration, RsEG146 induced cell death in the leaves of tobacco (Nicotiana tabacum 'Samsun'). The predicted signal peptide of RsEG146 was essential for its cell death-inducing activity, while the conserved enzymic active site was not required. The chitin-binding domain was critical for the cell death-inducing activity of RsEG146, with Gly47 identified as the key residue. Substitution of Gly47 with aspartate, glutamate, or proline significantly impaired the cell death-inducing activity of RsEG146. Additionally, transient and heterogeneous expression of RsEG146 enhanced the pathogenicity of Botrytis cinerea on tobacco, and silencing this gene through spray-induced gene silencing (SIGS) reduced the severity of the disease in maize, indicating that RsEG146 functions as an effector. Furthermore, RsEG146 triggered a plant immune response in tobacco. This study demonstrates that RsEG146 is a potential effector and plays a crucial role in the interactions between R. solani AG1 IA and its host.

Keywords: Rhizoctonia solani AG1 IA; cell death; glycoside hydrolase family 16; immune responses; spray‐induced gene silencing.

FIGURE 1

Sequence analysis and expression characters of RsEG146. (a) Total nucleotide sequence of RsEG146 amplified from Rhizoctonia solani AG1‐IA isolate YN‐7. The full‐length sequence of RsEG146 contains 1564 bp including nine exons and eight introns. (b) Conserved domain analysis of RsEG146 by using Conserved Domain Database. SP, signal peptide; ChtBD1, type 1 chitin‐binding domain; Lam G, laminin G domain; AS, active site. (c) Cladogram of glycoside hydrolase family 16 members in different anastomosis groups of R. solani and Ceratobasidium by using similar amino acid sequences of RsEG146. (d) Maize leaves inoculated with R. solani AG1 IA were collected 0, 12, 24, 36, 48 and 60 h post‐inoculation for detecting gene expression level by reverse transcription‐quantitative PCR. RsGAPDH expression was used as an internal reference gene. Column = mean ± SE (**p < 0.01).

FIGURE 2

Transiently expressed RsEG146 induced cell death and its subcelluar localisation in Nicotiana tabacum leaves. (a) Symptoms following transient expression of RsEG146 in N. tabacum at 36 and 48 h. (b) Trypan blue staining of N. tabacum leaves. (c) Conductivity of N. tabacum leaves. Transiently expressed GFP was used as a negative control. (d) Subcellular localisation of RsEG146 in N. tabacum leaves with endoplasmic reticulum localisation signal mCherry‐HDEL. The vector pMD1‐35S‐GFP carrying GFP was used as a control. Bar = 20 μm.

FIGURE 3

The signal peptide (SP) of RsEG146 is functional. (a) Functional validation of the SP of RsEG146 using yeast invertase secretion assay. All transformed YTK12 yeast strains grew on YPRAA medium with raffinose as the sole carbon source. α‐factor signal peptide from pPIC9K and N‐terminal sequence of Mg87 from Magnaporthe oryzae were used as positive and negative controls, respectively. The untransformed YTK12 did not grow on either CMD−W or YPRAA media, and the TTC solution remained colourless. Yeast growth on CMD−W medium was equally viable among the transformed strains. Mg87^SP: Negative control SP of Mg87 from M. oryzae ; α‐factor^SP: Positive control SP of α‐factor; RsEG146^SP: SPs of RsEG146. (b) Schematic presentation of RsEG146 and RsEG146^ΔSP. RsEG146^ΔSP: RsEG146 without SP. (c) Functional validation of the SP of RsEG146 by deleting the SP of RsEG146. The transient expression of RsEG146^ΔSP in Nicotiana tabacum maintained the leaf in a normal state without necrosis.

FIGURE 4

Cell death‐inducing activity of RsEG146 is independent of its enzymic activity. (a) Schematic presentation of RsEG146 and RsEG146^DAS. RsEG146^DAS: RsEG146 mutant with deleted active sites (DAS); EIDWE: Motif of RsEG146 enzymic active sites. *Indicates conserved amino acid residues. (b) The demonstration of relationships between enzymic activity and cell death‐inducing activity of RsEG146 by deleting enzymic active sites. The transient expression of RsEG146^DAS in Nicotiana tabacum also caused leaf necrosis.

FIGURE 5

Gly47 in chitin‐binding domain of RsEG146 is sufficient for inducing cell death in Nicotiana tabacum . (a) Schematic presentation and corresponding leaves of different RsEG146 domains that were used in agroinfiltration assays. Western blot analysis of RsEG146^C23‐70 production in tobacco. (b) Schematic presentation and corresponding leaves of different secondary structures of RsEG146^C23‐70 that were used in agroinfiltration assays. (c) Schematic presentation and corresponding leaves of different amino acids in RsEG146^C47‐53 that were used in agroinfiltration assays. All the experiments were replicated five times.

FIGURE 6

Necrosis function of Gly47 in RsEG146 can be incapacitated by acidic amino acid and proline substitution. (a) G47D, G47E and G47P did not induce cell death in Nicotiana tabacum . (b) Transient expression of GFP, wild type (WT), G47D, G47E and G47P in N. tabacum . (c) Protein electrostatic potential analysis of WT, G47D, G47E and G47P. The arrows indicates mutated sites. The mutations of G47D and G47E caused a change to negative charge (red), while G47P remained positively charge (blue).

FIGURE 7

RsEG146 played crucial roles to pathogenicity of Botrytis cinerea and Rhizoctonia solani. (a) Transiently expressed RsEG146 enhances the pathogenicity of B. cinerea on Nicotiana tabacum . RsEG146 was transiently expressed on lower leaves, followed by inoculating a mycelial block of B. cinerea on upper leaves after 24 h. (b) Heterogeneous expressed RsEG146 enhanced growth rate and pathogenicity of B. cinerea . (c) RsEG146 silenced by spray‐induced gene silencing does not affect mycelial growth but reduces pathogenicity of R. solani . Symptom, lesion size and RsEG146 expression level on R. solani ‐invaded Zea mays leaves. Column = mean ± SE (*p < 0.05, **p < 0.01).

FIGURE 8

RsEG146 triggers plant immunity response in Nicotiana tabacum . (a) Expression of four plant immunity‐related genes in N. tabacum leaves transiently expressing RsEG146. Column = mean ± SE (*p < 0.05, **p < 0.01). (b) Accumulation of reactive oxygen species (ROS) and H₂O₂ content in N. tabacum leaves transiently expressing RsEG146. These experiments were replicated three times with three leaves per biological replicate. (c) Deposition of callose in N. tabacum leaves transiently expressing RsEG146. (d) Variation of pH in N. tabacum leaves transiently expressing RsEG146. Column = mean ± SE (*p < 0.05).