Transcriptome analysis of sugarcane reveals differential switching of major defense signaling pathways in response to Sporisorium scitamineum isolates with varying virulent attributes.

Sugarcane smut caused by the basidiomycetous fungus Sporisorium scitamineum is one of the most devastating diseases that affect sugarcane production, globally. At present, the most practical and effective management strategy for the disease is the cultivation of resistant cultivars. In this connection, a detailed understanding of the host's defense mechanism in response to smut isolates with varying degrees of virulence at the molecular level would facilitate the development of reliable and durable smut-resistant sugarcane varieties. Hence, in this study, a comparative whole transcriptome analysis was performed employing Illumina RNA-seq in the smut susceptible cultivar Co 97009 inoculated with two distinct S. scitamineum isolates, Ss97009 (high-virulent) and SsV89101 (low-virulent) during the early phases of infection (2 dpi and 5 dpi) and at the phase of sporogenesis (whip emergence) (60 dpi). Though the differential gene expression profiling identified significant transcriptional changes during the early phase of infection in response to both the isolates, the number of differentially expressed genes (DEGs) were more abundant at 60 dpi during interaction with the high virulent isolate Ss97009, as compared to the low virulent isolate SsV89101. Functional analysis of these DEGs revealed that a majority of them were associated with hormone signaling and the synthesis of defense-related metabolites, suggesting a complex network of defense mechanisms is being operated in response to specific isolates of the smut pathogen. For instance, up-regulation of hormone-related genes, transcription factors, and flavonoid biosynthesis pathway genes was observed in response to both the isolates in the early phase of interaction. In comparison to early phases of infection, only a few pathogenesis-related proteins were up-regulated at 60 dpi in response to Ss97009, which might have rendered the host susceptible to infection. Strikingly, few other carbohydrate metabolism-associated genes like invertases were up-regulated in Ss97009 inoculated plants during the whip emergence stage, representing a shift from sucrose storage to smut symptoms. Altogether, this study established the major switching of defense signaling pathways in response to S. scitamineum isolates with different virulence attributes and provided novel insights into the molecular mechanisms of sugarcane-smut interaction.

Protoplast-mediated transformation in Sporisorium scitamineum facilitates visualization of in planta developmental stages in sugarcane.

BACKGROUND: Sporisorium scitamineum is the causative agent of smut disease in sugarcane. The tricky life cycle of S. scitamineum consists of three distinct growth stages: diploid teliospores, haploid sporidia and dikaryotic mycelia. Compatible haploid sporidia representing opposite mating types (MAT-1 and MAT-2) of the fungus fuse to form infective dikaryotic mycelia in the host tissues, leading to the development of a characteristic whip shaped sorus. In this study, the transition of distinct stages of in vitro life cycle and in planta developmental stages of S. scitamineum are presented by generating stable GFP transformants of S. scitamineum. METHODS AND RESULTS: Haploid sporidia were isolated from the teliospores of Ss97009, and the opposite mating types (MAT-1 and MAT-2) were identified by random mating assay and mating type-specific PCR. Both haploid sporidia were individually transformed with pNIIST plasmid, harboring an enhanced green fluorescent protein (eGFP) gene and hygromycin gene by a modified protoplast-based PEG-mediated transformation method. Thereafter, the distinct in vitro developmental stages including fusion of haploid sporidia and formation of dikaryotic mycelia expressing GFP were demonstrated. To visualize in planta colonization, transformed haploids (MAT-1gfp and MAT-2gfp) were fused and inoculated onto the smut susceptible sugarcane cultivar, Co 97009 and examined microscopically at different stages of colonization. GFP fluorescence-based analysis presented an extensive fungal colonization of the bud surface as well as inter- and intracellular colonization of the transformed S. scitamineum in sugarcane tissues during initial stages of disease development. Noticeably, the GFP-tagged S. scitamineum led to the emergence of smut whips, which established their pathogenicity, and demonstrated initial colonization, active sporogenesis and teliospore maturation stages. CONCLUSION: Overall, for the first time, an efficient protoplast-based transformation method was employed to depict clear-cut developmental stages in vitro and in planta using GFP-tagged strains for better understanding of S. scitamineum life cycle development.

Comparative expression analysis of potential pathogenicity-associated genes of high- and low-virulent Sporisorium scitamineum isolates during interaction with sugarcane.

Sporisorium scitamineum is a teleomorphic, biotrophic fungus causing the globally prevalent sugarcane smut disease in sugarcane. The severity of the disease depends on two major factors, viz. degree of resistance in the host genotype and virulence level of the pathogen. Hence, in this study, temporal transcriptomic expression of potential pathogenicity-associated genes of two distinctly virulent S. scitamineum isolates, viz. SsV89101 (low virulent) and Ss97009 (high virulent) were analyzed during interaction with a smut susceptible sugarcane cv. Co 97009 at six different time intervals. The pathogenicity-associated genes profiled in this study comprises 14 plant cell wall degrading enzymes (PCWDEs) and ten candidates secreted effector protein-coding (CSEPs) genes. Absolute quantification of pathogen biomass and comparative expression profiling analyses of these pathogenicity-associated genes during host-pathogen interaction indicated that there was a significant variation between low and high virulent isolates. More precisely, the higher and early expression (24 hpi) of certain PCWDEs, viz. Chitinase-1 and Laccase, and the CSEPs, viz. SUC2, SRT1 and CMU1 during the colonization of high virulent isolate suggested that they might possibly play a major role in facilitating faster and successful pathogen ingress, and tissue colonization than the less-virulent isolate. Transcript expression profiling of Chitinase and Laccases were also in correlation with their corresponding enzyme activity assays. Comprehensively, this quantitative temporal expression analysis has provided critical insights into the early expression of pathogenicity-associated genes and their putative role in attributing to higher virulence. Moreover, this study provides valuable clues for the screening of candidate virulence determinants for further functional characterization of the test pathogen isolates used for the evaluation of smut resistance in breeding clones. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02893-7.