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

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.

A small secreted protein, RsMf8HN, in Rhizoctonia solani triggers plant immune response, which interacts with rice OsHIPP28.

The necrotrophic phytopathogen Rhizoctonia solani (R. solani) causes disease in many plant species. This fungal genome encodes abundant small cysteine-rich (SCR)-secreted proteins in R. solani that may induce pathogenesis. To test their molecular functions, we introduced 10 SCR-secreted protein genes from R. solani into tobacco leaves via agroinfiltration. Consequently, we identified RsMf8HN, a novel SCR protein that triggers cell death and an oxidative burst in tobacco. RsMf8HN comprises 182 amino acids (aa), including a signal peptide (SP) of 17aa, and the protein has unique features: it is orthologous to an allergen protein Mal f 8 occurring in Malassezia species, and possesses a high glycine and serine content. RsMf8HN is coded in a genomic location along with its paralogues and a few other effector candidates. The elicitation of plant immunity by RsMf8HN was dependent on HSP90 and SGT1. RsMf8HN was translocated to multiple locations within the host cells: i.e., nuclei, chloroplasts, and plasma membranes. We confirmed the occurrence of in vivo cross-interactions of RsMf8HN with a rice molecule, the heavy metal-associated isoprenylated plant protein OsHIPP28, which is a protein related to the disease susceptibility factor Pi21. In summary, our results suggest that RsMf8HN is a potential effector that enables necrotrophic phytopathogens to interfere with host plant immunity.

A Novel, Small Cysteine-Rich Effector, RsSCR10 in Rhizoctonia solani Is Sufficient to Trigger Plant Cell Death.

The necrotrophic phytopathogen Rhizoctonia solani (R. solani) is a fungus that causes disease in a wide range of plant species. Fungal genomes encode abundant, small cysteine-rich (SCR) secreted proteins, and the probable importance of these to pathogenesis has been highlighted in various pathogens. However, there are currently no reports of an R. solani SCR-secreted protein with evidential elicitor activity. In this study, the molecular function of 10 SCR-secreted protein genes from R. solani was explored by agroinfiltration into Nicotiana benthamiana (N. benthamiana) leaves, and a novel SCR protein RsSCR10 was identified that triggered cell death and oxidative burst in tobacco. RsSCR10 comprises 84 amino acids, including a signal peptide (SP) of 19 amino acids that is necessary for RsSCR10 to induce tobacco cell death. Elicitation of cell death by RsSCR10 was dependent on Hsp90 but not on RAR1, proving its effector activity. Two cysteine residues have important effects on the function of RsSCR10 in inducing cell death. Furthermore, RsSCR10 showed cross-interaction with five rice molecules, and the inferred functions of these rice proteins suggest they are instrumental in how the host copes with adversity. Overall, this study demonstrates that RsSCR10 is a potential effector that has a critical role in R. solani AG1 IA-host interactions.

Comparative Mitogenomic Analysis and the Evolution of Rhizoctonia solani Anastomosis Groups.

Mitochondria are the major energy source for cell functions. However, for the plant fungal pathogens, mitogenome variations and their roles during the host infection processes remain largely unknown. Rhizoctonia solani, an important soil-borne pathogen, forms different anastomosis groups (AGs) and adapts to a broad range of hosts in nature. Here, we reported three complete mitogenomes of AG1-IA RSIA1, AG1-IB RSIB1, and AG1-IC, and performed a comparative analysis with nine published Rhizoctonia mitogenomes (AG1-IA XN, AG1-IB 7/3/14, AG3, AG4, and five Rhizoctonia sp. mitogenomes). These mitogenomes encoded 15 typical proteins (cox1-3, cob, atp6, atp8-9, nad1-6, nad4L, and rps3) and several LAGLIDADG/GIY-YIG endonucleases with sizes ranging from 109,017 bp (Rhizoctonia sp. SM) to 235,849 bp (AG3). We found that their large sizes were mainly contributed by repeat sequences and genes encoding endonucleases. We identified the complete sequence of the rps3 gene in 10 Rhizoctonia mitogenomes, which contained 14 positively selected sites. Moreover, we inferred a robust maximum-likelihood phylogeny of 32 Basidiomycota mitogenomes, representing that seven R. solani and other five Rhizoctonia sp. lineages formed two parallel branches in Agaricomycotina. The comparative analysis showed that mitogenomes of Basidiomycota pathogens had high GC content and mitogenomes of R. solani had high repeat content. Compared to other strains, the AG1-IC strain had low substitution rates, which may affect its mitochondrial phylogenetic placement in the R. solani clade. Additionally, with the published RNA-seq data, we investigated gene expression patterns from different AGs during host infection stages. The expressed genes from AG1-IA (host: rice) and AG3 (host: potato) mainly formed four groups by k-mean partitioning analysis. However, conserved genes represented varied expression patterns, and only the patterns of rps3-nad2 and nad1-m3g18/mag28 (an LAGLIDADG endonuclease) were conserved in AG1-IA and AG3 as shown by the correlation coefficient analysis, suggesting regulation of gene repertoires adapting to infect varied hosts. The results of variations in mitogenome characteristics and the gene substitution rates and expression patterns may provide insights into the evolution of R. solani mitogenomes.

Identification of the Novel Effector RsIA_NP8 in Rhizoctonia solani AG1 IA That Induces Cell Death and Triggers Defense Responses in Non-Host Plants.

Rhizoctonia solani AG1 IA is a necrotrophic fungus that causes rice sheath blight, one of the most significant rice diseases in the world. However, little is known about the pathogenic mechanisms and functions of effectors in R. solani AG1 IA. We performed functional studies on effectors in R. solani AG1 IA and found that, of 11 putative effectors tested, only RsIA_NP8 caused necrosis in the leaves of Nicotiana benthamiana. The predicted signal peptide of this protein was required to induce cell death, whereas predicted N-glycosylation sites were not required. RsIA_NP8 was upregulated during early infection, and the encoded protein was secreted. Furthermore, the ability of RsIA_NP8 to trigger cell death in N. benthamiana depended on suppressor of G2 allele of Skp1 (SGT1) and heat shock protein 90 (HSP90), but not on Mla12 resistance (RAR1) and somatic embryogenesis receptor-like kinase (SERK3). A natural variation that prevents the triggering of cell death in N. benthamiana was found in RsIA_NP8 in 25 R. solani AG1 IA strains. It is important to note that RsIA_NP8 induced the immune response in N. benthamiana leaves. Collectively, these results show that RsIA_NP8 is a possible effector that plays a key role in R. solani AG1 IA-host interactions.

Comparison of gene co-networks analysis provide a systems view of rice (Oryza sativa L.) response to Tilletia horrida infection.

The biotrophic soil-borne fungus Tilletia horrida causes rice kernel smut, an important disease affecting the production of rice male sterile lines in most hybrid rice growing regions of the world. There are no successful ways of controlling this disease and there has been little study of mechanisms of resistance to T. horrida. Based on transcriptional data of different infection time points, we found 23, 782 and 23, 718 differentially expressed genes (fragments per kilobase of transcript sequence per million, FPKM >1) in Jiangcheng 3A (resistant to T. horrida) and 9311A (susceptible to T. horrida), respectively. In order to illuminate the differential responses of the two rice male sterile lines to T. horrida, we identified gene co-expression modules using the method of weighted gene co-expression network analysis (WGCNA) and compared the different biological functions of gene co-expression networks in key modules at different infection time points. The results indicated that gene co-expression networks in the two rice genotypes were different and that genes contained in some modules of the two groups may play important roles in resistance to T. horrida, such as DTH8 and OsHop/Sti1a. Furthermore, these results provide a global view of the responses of two different phenotypes to T. horrida, and assist our understanding of the regulation of expression changes after T. horrida infection.