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.

Transcriptional reprogramming of major defense-signaling pathways during defense priming and sugarcane-Colletotrichum falcatum interaction.

Red rot caused by Colletotrichum falcatum poses a serious threat to sugarcane cultivation in many tropical and sub-tropical countries. Deciphering the molecular network of major defense-signaling pathways in sugarcane cultivars with varying red rot resistance is essential to elucidate the phenomenon of defense priming exerted by resistance inducers. Therefore, in this study, expression pattern of transcripts coding for major defense-signaling pathway regulatory genes was profiled during compatible and incompatible interactions and in response to defense priming using qRT-PCR. Candidate genes that were profiled are involved in or related to hypersensitive response and reactive oxygen species production (HR/ROS), salicylic acid (SA), and jasmonic acid/ethylene (JA/ET) pathways. For compatible and incompatible interactions, susceptible (CoC 671), field tolerant (Co 86032) and resistant (Co 93009) sugarcane cultivars were used, whereas for defense priming, benzothiadiazole (BTH) and the pathogen-associated molecular patterns (PAMPs) of C. falcatum viz., CfEPL1 (eliciting plant response-like) and CfPDIP1 (plant defense inducing protein) were used in CoC 671 cultivar. Results indicated that the master regulator of defense pathways, nonexpressor of pathogenesis-related genes 1 (NPR1) was highly upregulated in incompatible interactions (in both Co 86032 and Co 93009) than the compatible interaction along with SA pathway-associated genes. Similarly, in response to defense priming with BTH, CfEPL1 and CfPDIP1, only the SA pathway-associated genes showed considerable upregulation at 0 h post inoculation (hpi) and other intermittent time points. Overall, this study showed that SA-mediated defense pathway is the most predominant pathway reprogrammed during priming with BTH, CfEPL1 and CfPDIP1 and substantiated the earlier findings that these agents indeed induce systemic resistance against red rot of sugarcane.

CfPDIP1, a novel secreted protein of Colletotrichum falcatum, elicits defense responses in sugarcane and triggers hypersensitive response in tobacco.

Colletotrichum falcatum, a hemibiotrophic fungal pathogen, causes one of the major devastating diseases of sugarcane-red rot. C. falcatum secretes a plethora of molecular signatures that might play a crucial role during its interaction with sugarcane. Here, we report the purification and characterization of a novel secreted protein of C. falcatum that elicits defense responses in sugarcane and triggers hypersensitive response (HR) in tobacco. The novel protein purified from the culture filtrate of C. falcatum was identified by MALDI TOF/TOF MS and designated as C. falcatum plant defense-inducing protein 1 (CfPDIP1). Temporal transcriptional profiling showed that the level of CfPDIP1 expression was greater in incompatible interaction than the compatible interaction until 120 h post-inoculation (hpi). EffectorP, an in silico tool, has predicted CfPDIP1 as a potential effector. Functional characterization of full length and two other domain deletional variants (CfPDIP1ΔN1-21 and CfPDIP1ΔN1-45) of recombinant CfPDIP1 proteins has indicated that CfPDIP1ΔN1-21 variant elicited rapid alkalinization and induced a relatively higher production of hydrogen peroxide (H2O2) in sugarcane suspension culture. However, in Nicotiana tabacum, all the three forms of recombinant CfPDIP1 proteins triggered HR along with the induction of H2O2 production and callose deposition. Further characterization using detached leaf bioassay in sugarcane revealed that foliar priming with CfPDIP1∆1-21 has suppressed the extent of lesion development, even though the co-infiltration of CfPDIP1∆1-21 with C. falcatum on unprimed leaves increased the extent of lesion development than control. Besides, the foliar priming has induced systemic expression of major defense-related genes with the concomitant reduction of pathogen biomass and thereby suppression of red rot severity in sugarcane. Comprehensively, the results have suggested that the novel protein, CfPDIP1, has the potential to trigger a multitude of defense responses in sugarcane and tobacco upon priming and might play a potential role during plant-pathogen interactions.

Comparative secretome analysis of Colletotrichum falcatum identifies a cerato-platanin protein (EPL1) as a potential pathogen-associated molecular pattern (PAMP) inducing systemic resistance in sugarcane.

Colletotrichum falcatum, an intriguing hemibiotrophic fungal pathogen causes red rot, a devastating disease of sugarcane. Repeated in vitro subculturing of C. falcatum under dark condition alters morphology and reduces virulence of the culture. Hitherto, no information is available on this phenomenon at molecular level. In this study, the in vitro secretome of C. falcatum cultured under light and dark conditions was analyzed using 2-DE coupled with MALDI TOF/TOF MS. Comparative analysis identified nine differentially abundant proteins. Among them, seven proteins were less abundant in the dark-cultured C. falcatum, wherein only two protein species of a cerato-platanin protein called EPL1 (eliciting plant response-like protein) were found to be highly abundant. Transcriptional expression of candidate high abundant proteins was profiled during host-pathogen interaction using qRT-PCR. Comprehensively, this comparative secretome analysis identified five putative effectors, two pathogenicity-related proteins and one pathogen-associated molecular pattern (PAMP) of C. falcatum. Functional characterization of three distinct domains of the PAMP (EPL1) showed that the major cerato-platanin domain (EPL1∆N1-92) is exclusively essential for inducing defense and hypersensitive response (HR) in sugarcane and tobacco, respectively. Further, priming with EPL1∆N1-92 protein induced systemic resistance and significantly suppressed the red rot severity in sugarcane. BIOLOGICAL SIGNIFICANCE: Being the first secretomic investigation of C. falcatum, this study has identified five potential effectors, two pathogenicity-related proteins and a PAMP. Although many reports have highlighted the influence of light on pathogenicity, this study has established a direct link between light and expression of effectors, for the first time. This study has presented the influence of a novel N-terminal domain of EPL1 in physical and biological properties and established the functional role of major cerato-platanin domain of EPL1 as a potential elicitor inducing systemic resistance in sugarcane. Comprehensively, the study has identified proteins that putatively contribute to virulence of C. falcatum and for the first time, demonstrated the potential role of EPL1 in inducing PAMP-triggered immunity (PTI) in sugarcane.

In vitro secretomic analysis identifies putative pathogenicity-related proteins of Sporisorium scitamineum - The sugarcane smut fungus.

Sporisorium scitamineum, the sugarcane smut pathogen, relies predominantly on its secretome to successfully colonise its host, in accordance with other related smut fungi. Considering the significance of deciphering its secretome, we have examined alterations in the in vitro secretome of S. scitamineum in response to synthetic and sugarcane meristem tissue-amended growth media, so as to identify host signal responsive secretory proteins. Secretory proteins that were differentially abundant and exclusively secreted in response to host extract media were identified by two-dimensional gel electrophoresis coupled with MALDI-TOF/TOF MS. Of the 16 differentially abundant and exclusively secreted proteins, nine proteins were identified. Among which, six were related to cell wall modification, morphogenesis, polysaccharide degradation, and carbohydrate metabolism. In planta gene expression profiling indicated that five in vitro secreted proteins were expressed in distinct patterns by S. scitamineum during different stages of infection with relatively higher expression at 1 day after inoculation, suggesting that these proteins could be aiding S. scitamineum at early time points in penetration and colonisation of sugarcane cells. The present study has provided insights into the alterations occurring in the secretome of S. scitamineum at in vitro conditions and has resulted in the identification of secretory proteins that are possibly associated with pathogenicity of the sugarcane smut fungus.

Proteomic analysis of a compatible interaction between sugarcane and Sporisorium scitamineum.

Smut caused by Sporisorium scitamineum is one of the important diseases of sugarcane with global significance. Despite the intriguing nature of sugarcane, S. scitamineum interaction, several pertinent aspects remain unexplored. This study investigates the proteome level alterations occurring in the meristem of a S. scitamineum infected susceptible sugarcane cultivar at whip emergence stage. Differentially abundant proteins were identified by 2DE coupled with MALDI-TOF/TOF-MS. Comprehensively, 53 sugarcane proteins identified were related to defence, stress, metabolism, protein folding, energy, and cell division; in addition, a putative effector of S. scitamineum, chorismate mutase, was identified. Transcript expression vis-à-vis the activity of phenylalanine ammonia lyase was relatively higher in the infected meristem. Abundance of seven candidate proteins in 2D gel profiles was in correlation with its corresponding transcript expression levels as validated by qRT-PCR. Furthermore, this study has opened up new perspectives on the interaction between sugarcane and S. scitamineum.

Proteome readjustments in the apoplastic space of Arabidopsis thaliana ggt1 mutant leaves exposed to UV-B radiation.

Ultraviolet-B radiation acts as an environmental stimulus, but in high doses it has detrimental effects on plant metabolism. Plasma membranes represent a major target for Reactive Oxygen Species (ROS) generated by this harmful radiation. Oxidative reactions occurring in the apoplastic space are counteracted by antioxidative systems mainly involving ascorbate and, to some extent, glutathione. The occurrence of the latter and its exact role in the extracellular space are not well documented, however. In Arabidopsis thaliana, the gamma-glutamyl transferase isoform (GGT1) bound to the cell wall takes part in the so-called gamma-glutamyl cycle for extracellular glutathione degradation and recovery, and may be implicated in redox sensing and balance. In this work, oxidative conditions were imposed with Ultraviolet-B radiation (UV-B) and studied in redox altered ggt1 mutants. The response of ggt1 knockout Arabidopsis leaves to UV-B radiation was assessed by investigating changes in extracellular glutathione and ascorbate content and their redox state, and in apoplastic protein composition. Our results show that, on UV-B exposure, soluble antioxidants respond to the oxidative conditions in both genotypes. Rearrangements occur in their apoplastic protein composition, suggesting an involvement of Hydrogen Peroxide (H2O2), which may ultimately act as a signal. Other important changes relating to hormonal effects, cell wall remodeling, and redox activities are discussed. We argue that oxidative stress conditions imposed by UV-B and disruption of the gamma-glutamyl cycle result in similar stress-induced responses, to some degree at least. Data are available via ProteomeXchange with identifier PXD001807.

Sugarcane proteomics: An update on current status, challenges, and future prospects.

Sugarcane is one of the most important commercial crops cultivated worldwide for the production of crystal sugar, ethanol, and other related by-products. Unlike other comparable monocots like sorghum, maize, and rice, sugarcane genome by virtue of its polyploidy nature remains yet to be fully deciphered. Proteomics-an established complementary tool to genomics is at its infancy in sugarcane as compared to the other monocots. However, with the surge in genomics research accomplished by next-generation sequencing platforms, sugarcane proteomics has gained momentum. This review summarizes the available literature from 1970 to 2014, which ensures a comprehensive coverage on sugarcane proteomics-a topic first of its kind to be reviewed. We herewith compiled substantial contributions in different areas of sugarcane proteomics, which include abiotic and biotic stresses, cell wall, organelle, and structural proteomics. The past decade has witnessed a paradigm shift in the pace with which sugarcane proteomics is progressing, as evident by the number of research publications. In addition to extensively reviewing the progress made thus far, we intend to highlight the scope in sugarcane proteomics, with an aspiration to instigate focused research on sugarcane to harness its full potential for the human welfare.