Comparative secretome analysis of different smut fungi and identification of plant cell death-inducing secreted proteins from Tilletia horrida

Abstract

Background: Tilletia horrida is a basidiomycete fungus that causes rice kernel smut, one of the most important rice diseases in hybrid rice growing areas worldwide. However, little is known about its mechanisms of pathogenicity. We previously reported the genome of T. horrida, and 597 genes that encoded secreted proteins were annotated. Among these were some important effector genes related to pathogenicity.

Results: A secretome analysis suggested that five Tilletia fungi shared more gene families than were found in other smuts, and there was high conservation between them. Furthermore, we screened 597 secreted proteins from the T. horrida genome, some of which induced expression in host-pathogen interaction processes. Through transient expression, we demonstrated that two putative effectors could induce necrosis phenotypes in Nicotiana benthamiana. These two encoded genes were up-regulated during early infection, and the encoded proteins were confirmed to be secreted using a yeast secretion system. For the putative effector gene smut_5844, a signal peptide was required to induce non-host cell death, whereas ribonuclease catalytic active sites were required for smut_2965. Moreover, both putative effectors could induce an immune response in N. benthamiana leaves. Interestingly, one of the identified potential host interactors of smut_5844 was laccase-10 protein (OsLAC10), which has been predicted to be involved in plant lignification and iron metabolism.

Conclusions: Overall, this study identified two secreted proteins in T. horrida that induce cell death or are involved in defense machinery in non-host plants. This research provides a useful foundation for understanding the interaction between rice and T. horrida.

Keywords: Cell-death; Effector proteins; RNase active site; Signal peptides; Tilletia horrida; Yeast two-hybrid.

Fig. 1

Homology analysis of Tilletia horrida secreted proteins with other smut fungi. a Venn diagram showing orthologs between the nine smut fungi secreted proteins. b Venn diagram showing orthologs between the secreted proteins of the five different smut fungus genera. c Venn diagram showing orthologs between the five Tilletia fungi secreted proteins. tca denotes Tilletia caries; tco denotes Tilletia controversa; tin denotes Tilletia indica; twa denotes Tilletia walkeri; tho denotes Tilletia horrida; sre denotes Sporisorium reilianum; ssc denotes Sporisorium scitamineum; uma denotes Ustilago maydis; uho denotes Ustilago hordei

Fig. 2

The phylogeny of the 131 putative effectors in T. horrida

Fig. 3

The putative effectors in T. horrida induce necrotic cell-death phenotypes in Nicotiana benthamiana and treated leaves were stained with Trypan blue. a The symptoms of N. benthamiana leaves after inoculation with smut_2965 and smut_5844. PMDC32-GFP means PMDC32 vector containing the green fluorescent protein (GFP) gene; the same labeling was used for PMDC32-BAX, PMDC32–2965, and PMDC32–5844. Numbers, e.g., 23/27, indicate that 23 of 27 infiltrated leaves exhibited cell death or mottling phenotypes. Agrobacterium carrying green fluorescent protein (GFP) served as a negative control. Agrobacterium carrying the BAX vector served as a positive control. Representative photos were taken four days after infiltration. b The expressed proteins were detected in the leaves of N. benthamiana by western blot experiments. M: marker; B1–4: smut_2965 (13.86 kDa), smut_5844 (24.84 kDa), BAX (21 kDa), and GFP (27 kDa). c The phylogeny of smut_2965 and 12 fungi homology secreted proteins. The phylogeny was constructed using Mega v7.0.26. tca denotes Tilletia caries, tco denotes Tilletia controversa, tin denotes Tilletia indica, twa denotes Tilletia walkeri, tho denotes Tilletia horrida, uho denotes Ustilago hordei, uma denotes Ustilago maydis, src denotes Sporisorium reilianum, ssc denotes Sporisorium scitamineum, lbi denotes Laccaria bicolor, sco denotes Schizophyllum commune, mgr denotes Magnaporthe grisea, uvi denotes Ustilaginoidea virens, and rso denotes Rhizoctonia solani

Fig. 4

Trend analysis of putative effector genes. a The expression of 12 genes in profile 18 (smut_5844 was included) and 14 genes in profile 19 (smut_2965 was included). b The 291 differentially expressed genes that encoded secreted proteins were clustered in 23 profiles (no genes were found in profile 12). In the analysis, smut_3800 was clustered in profile 18

Fig. 5

Functional validation of signal peptides (SPs) of putative T. horrida effectors using a yeast invertase secretion assay. The SPs predicted two T. horrida putative effectors. All transformed yeast strains YTK12 were able to grow on YPRAA media with raffinose as the sole carbon source (1% yeast extract, 2% peptone, 2% raffinose, and 2 μg of antimycin A per liter). The N-terminal sequences of Phytophthora sojae Avr1b and Magnaporthe oryzae Mg87 were used as positive and negative controls, respectively. The untransformed YTK12 did not grow on either CMD-W (0.67% yeast N base without amino acids, 0.075% tryptophan dropout supplement, 2% sucrose, 0.1% glucose, and 2% agar) or YPRAA media. Yeast growth on CMD-W media was equally viable among the transformed strains. Row a: CMD-W media; row b: YPRAA media; Mg87 (−): negative controls Mg87 SPs; Avr1b (+):positive controls Avr1b SPs; sp2965: SPs of smut_2965; sp5844: SPs of smut_5844

Fig. 6

a The ability of truncated T. horrida secreted proteins lacking signal peptides (SPs) to induced necrotic cell-death. PMDC32–2965-sp denotes smut_2965 without SPs, PMDC32–5844-sp denote smut_5844 without SPs. Numbers, e.g., 20/20, indicate that 20 of 20 infiltrated leaves exhibited cell death or mottling phenotypes. b Expressed proteins were detected in the leaves of N. benthamiana following western blot experiments. M: marker; b1–2: smut_2965-sp (12.9 kDa) and smut_5844-sp (24 kDa)

Fig. 7

The predicted RNase active site of smut_2965 is required for its cell-death triggering ability. a The mutant proteins smut_2965–60, smut_2965–79, smut_2965–96, and smut_2965–112 lost the ability to induce cell-death, while smut_5844–68 and smut_5844–174 induced cell-death in Nicotiana benthamiana leaves. GFP refers to the PMDC32 vector containing the green fluorescent protein (GFP) gene, the same labeling is used for 2965, 5844, 2965∆60, 2965∆79, 2965∆96, 2965∆112, 5844∆68, and 5844∆147. Numbers, e.g., 0/35, indicate that 0 of 35 infiltrated leaves exhibiting cell-death or mottling phenotypes. b Protein expression of the smut_2965 and smut_5844 mutant proteins in the infiltrated leaves was detected by western blotting. M:marker; b1–6: smut_2965∆60 (13.74 kDa), smut_2965∆79 (13.74 kDa), smut_2965∆96 (13.74 kDa), smut_2965∆112 (13.74 kDa), smut_5844∆68 (24.72 kDa), and smut_5844∆147 (24.72 kDa)

Fig. 8

Expression profiles of two putative effector genes during T. horrida infection of the kernel smut–resistant and -susceptible rice male sterile lines. The T. horrida–inoculated panicles of the kernel smut–resistant cultivar Jiangcheng 3A (resistant rice male sterile line, R) and susceptible cultivar 9311A (susceptible rice male sterile line, S) were collected at 0, 8, 12, 24, 48, and 72 h post-inoculation for gene expression analyses using quantitative real time reverse transcription-polymerase chain reaction (qRT-PCR) assay. The UBQ gene was used to normalize the data, which are presented as normalized relative quantities scaled to the control. Error bars indicated the standard deviation of five independent replicates. For statistical analysis, we used analysis of variance with the R-package “ggpubr”, and significance was set at P < 0.05. *: P < 0.05, **: P < 0.01, ***: P < 0.001

Fig. 9

a Gene expression levels of related to plant immunity in Nicotiana benthamiana leaves transiently expressing two effector genes for 8, 12, 24, 48, and 72 h. Nicotiana benthamiana leaves were infiltrated with Agrobacterium tumefaciens GV3101 carrying either the PMDC32 empty (Control) or PMDC32-smut_5844 and smut_2965 vector. The plants were incubated in a plant growth room with 12 h/12 h, night/day photoperiods at 20–24 °C with 60% relative humidity. The leaves were harvested after 8, 12, 24, 48, and 72 h post-infiltration. Total RNA was extracted and reverse transcribed into cDNA. The qPCR was performed on the cDNA using specific primers for several genes related to activation of plant immunity. Actin gene was used to normalize the data, which are presented as normalized relative quantities scaled to control. Error bars indicated the standard deviation of 3 independent replicates. b N. benthamiana leaf that infected with agrobacterium carrying GFP was dyed used DAB. c N. benthamiana leaf that infected with agrobacterium carrying smut_2965 was dyed used DAB. d N. benthamiana leaf that infected with agrobacterium carrying smut_5844 was dyed used DAB. For statistical analysis, we used analysis of variance with the R-package “ggpubr”, and significance was set at P < 0.05. *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001

Fig. 10

Yeast two-hybrid analysis of the smut_5844. a-b Autoactivation activity test of smut_5844 protein in yeast. a: SD-Trp/−Leu, b: SD-Trp/−Leu/−His/−Ade/X-α-gal. pAD-WT/pBD-WT was used as positive controls, and pAD-WT/pLaminC was used as a negative control. WT, Wild-type fragment C of lambda cI repressor (aa 132–236); Mut, E233K mutated fragment of lambda cl repressor (aa 132–236); Lamin C, human lamin C (aa 67–230). c Interaction between smut_5844 and OsLAC10 in a yeast two-hybrid system. P53 and SV40 were used as a pair of positive control. Lam and SV40 were used as a negative control. SD-2, SD-Trp-Leu; SD-4, SD-Trp-Leu-His-Ade. d The expression patterns of OsLAC10 and OsRbs5 in resistant and susceptible rice male sterile lines after T. horrida inoculation