Differential host responses of sugarcane to Colletotrichum falcatum reveal activation of probable effector triggered immunity (ETI) in defence responses.

KEY MESSAGE: The differential compatibility responses of sugarcane to Colletotrichum falcatum pathotypes depend on the nature of both host primary defence signalling cascades and pathogen virulence. The complex polyploidy of sugarcane genome and genetic variations in different cultivars of sugarcane remain a challenge to identify and characterise specific genes controlling the compatible and incompatible interactions between sugarcane and the red rot pathogen, Colletotrichum falcatum. To avoid host background variation in the interaction study, suppression subtractive hybridization (SSH)-based next-generation sequencing (NGS) technology was used in a sugarcane cultivar Co 7805 which is compatible with one C. falcatum pathotype but incompatible with another one. In the incompatible interaction (ICI-less virulent) 10,038 contigs were assembled from ~ 54,699,263 raw reads, while 4022 contigs were assembled from ~ 52,509,239 in the compatible interaction (CI-virulent). The transcripts homologous to CEBiP receptor and those involved in the signalling pathways of ROS, Ca2+, BR, and ABA were expressed in both interaction responses. In contrast, MAPK, ET, PI signalling pathways and JA amino conjugation related transcripts were found only in ICI. In temporal gene expression assays, 16 transcripts showed their highest induction in ICI than CI. Further, more than 17 transcripts specific to the pathogen were found only in CI, indicating that the pathogen colonizes the host tissue whereas it failed to do so in ICI. Overall, this study has identified for the first time that a probable PAMP triggered immunity (PTI) in both responses, while a more efficient effector triggered immunity (ETI) was found only in ICI. Moreover, pathogen proliferation could be predicted in CI based on transcript expression, which were homologous to Glomerella graminicola, the nearest clade to the perfect stage of C. falcatum (G. tucumanensis).

Carbohydrate active enzymes (CAZy) regulate cellulolytic and pectinolytic enzymes in Colletotrichum falcatum causing red rot in sugarcane.

Colletotrichum falcatum, an ascomycete pathogen causes red rot of sugarcane which is specialized to infect cane stalks. Cellulolytic and pectinolytic enzymes are necessary for degradation of plant cell wall which stands as barrier for successful fungal pathogenesis. In the study, we have confined to the CAZy genes that regulate cellulolytic and pectinolytic enzymes in two distinctive pathotypes of C. falcatum. Comparative transcriptome analysis revealed that a number of CAZy genes producing cellulolytic and pectinolytic enzyme were present in the virulent (Cf671) and least virulent (RoC) pathotypes. Two consecutive transcriptome analyses (in vitro) were performed using Illumina Hi Seq 2500, further analysis was done with various bioinformatic tools. In vitro expression analysis of cutinase, glycoside hydrolyase and pectin-related genes revealed number of genes that attributes virulence. Numerous pectin-related genes involved in degradation of plant cell wall, pectinase and pectin lyase are considered to be key precursor in degradation of pectin in sugarcane. These results suggest that cellulolytic enzymes, cutinase and pectin-related genes are essential for degradation of sugarcane cell wall and considered to be an important pathogenic factor in C. falcatum. This is the first detailed report on sugarcane cell wall-degrading enzymes during its interaction with C. falcatum and also this comparative transcriptome analysis provided more insights into pathogen mechanism on C. falcatum. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-022-03113-6.

Knock-down of glucose transporter and sucrose non-fermenting gene in the hemibiotrophic fungus Colletotrichum falcatum causing sugarcane red rot.

Red rot caused by Colletotrichum falcatum, is one of the economically important disease of sugarcane and breeding for resistant varieties is considered to be the major solution to manage the disease. However, breakdown of red rot resistance become usual phenomenon due to development of newer races by culture adaptation on newly released varieties. Hence it is needed to characterize the genes responsible for pathogen virulence in order to take care of host resistance or to manage the disease by other methods. The transcript studies gave foundation to characterize the huge number of pathogenicity determinants and their role in pathogenesis. Here we studied role of two important genes viz., Glucose Transporter (GT) and Sucrose Non-Fermenting1 (SNF1) during pathogenesis of C. falcatum, which said to be involved in carbon source metabolism. Sugar metabolism has a vital role in disease progression of C. falcatum by regulating their cell growth, metabolism and development of the pathogen during various stages of infection. The present study was aimed to find out the role of GT and SNF1 genes in response to pathogenicity by RNA silencing (RNAi) approach. Knock-down of the target pathogenicity gene homologs in standard C. falcatum isolate Cf671 was carried out by amplifying sense and antisense fragments of targets individually using pSilent-1 vector. The expression cassette was cloned into the binary vector pCAMBIA1300 followed by fungal transformation through Agarobacterium mediated transformation. Resulted mutants of both the genes showed less virulence compared to wild type isolate. Simultaneously, both the mutants did not produce spores. Moreover, the molecular confirmation of the mutants displayed the expression of hygromycin gene with reduced expression of the target gene during host-pathogen interaction. Knockdown of the pathogenicity related genes (GT and SNF1) by RNAi approach corroborate the possible role of the genes in causing the disease.

Development and characterization of genomic SSR marker for virulent strain-specific Colletotrichum falcatum infecting sugarcane.

Colle totrichum falcatum, an intriguing pathogen causing red rot in sugarcane, exhibits enormous variation for pathogenicity under field conditions. A species-specific marker is very much needed to classify the virulence among the varying population and to identify the potential of a pathotype by mining the microsatellites, which are considered to be the largest genetic source to develop molecular markers for an organism. In this study, we have mined the C. falcatum genome using MISA database which yielded 12,121 SSRs from 48.1 Mb and 2745 SSRs containing sequences. The most frequent SSR types from the genome of C. falcatum was di-nucleotide which constitutes 50.89% followed by tri-nucleotide 39.60%, hepta-nucleotide 6.7%, hexa-nucleotide 1.38% and penta-nucleotide 1.3%. Over 90 SSR containing sequences from the genome were predicted using BlastX which are found to be non-homologs. Most of the annotated SSR containing sequences fell in CAZy superfamilies, proteases, peptidases, plant cell wall degrading enzymes (PCDWE) and membrane transporters which are considered to be pathogenicity gene clusters. Among them, glycosyl hydrolases (GH) were found to be abundant in SSR containing sequences which again proved our previous transcriptome results. Our in-silico results suggested that the mined microsatellites from C. falcatum genome show absence of homolog sequences which suggests that these markers could be used as an ideal species-specific molecular marker. Two virulence specific markers were characterized using conventional PCR assays from C. falcatum along with virulent species-specific (VSS) marker developed for C. gloeosporioides. The study lays the foundation for the development of C. falcatum specific molecular marker to phenotype the pathotypes based on virulence.

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.

Differential regulation of defense-related gene expression in response to red rot pathogen Colletotrichum falcatum infection in sugarcane.

Red rot is a serious disease of sugarcane caused by the fungus Colletotrichum falcatum imposing a considerable economic loss annually in all sugarcane-producing countries. In this study, we analyzed the early resistance response of sugarcane to red rot fungus by comparing the differences between control and inoculated stalk tissues. Differential display reverse transcription polymerase chain reaction (DD-RT-PCR) was employed to identify altered expression of genes in disease-resistant cv Co 93009, in response to pathogen infection. DD-RT-PCR identified 300 differentially expressed transcripts of which 112 were selected for further analysis. Cloning and sequence analysis of the isolated cDNA fragments resulted in functional categorization of these clones into five categories, of which the defense/stress/signaling group was the largest, with clones homologous to genes known to be actively involved in various pathogenesis-related functions in plant species. This group showed overexpression of several transcripts related to ethylene-mediated and jasmonic acid pathway of plant defense mechanisms. Of the 112 expressed sequence tags, validation of expression was carried out for five important genes whose role in plant defense mechanisms is well established. This is the first report of Colletotrichum-mediated gene regulation in sugarcane which has provided a set of candidate genes for detailed molecular dissection of signaling and defense responses in tropical sugarcane during the onset of red rot resistance.