Sugarcane Defense Research Articles

Two putative calcium-dependent protein kinases are involved in the regulation of sugarcane defense genes

Sugarcane is a primary sugar crop and an important source of bioenergy. Pathogens are the major factors affecting sugarcane yield and sugar content. However, the mechanisms of sugarcane defense regulation remain largely unknown, and research on prospective genetic targets for modification is scarce. As the main class of calcium sensors, calcium-dependent protein kinases (CDPKs/CPKs) play a crucial role in the immune regulatory network. Using sugarcane genomic data, we identified 229 putative ScCDPKs in primordial specie Saccharum officinarum. Searching the putative CDPKs in sugarcane cultivars from National Center for Biotechnology Information (NCBI), 12 putative ScCDPKs in cultivars were identified. Phylogenetic analysis revealed evolutionary relationships among these CDPKs in sugarcane cultivars and those in S. officinarum, Arabidopsis thaliana, and rice. Truncation mutants of ScCDPKs were introduced into the reporter system and examined for activity in inducing Sugarcane Pathogenesis Related Protein 1 (ScPR1). The results showed that truncated ScCDPK1 and ScCDPK8 induce higher expression of ScPR1 than full-length ScCDPK1 and ScCDPK8. Additionally, transient expression of truncated ScCDPK1 and ScCDPK8 exhibit stronger activity in sugarcane protoplasts for activation of ScPR1 and sugarcane Serine Protease Inhibitor (ScSPI). These results demonstrate that ScCDPK1 and ScCDPK8 possess auto-inhibitory activity. The findings of this study provide a basis for an in-depth study of the sugarcane CDPK gene family and lay the foundation for further genetic improvement.

Insights into Reactive Oxygen Species Production-Scavenging System Involved in Sugarcane Response to Xanthomonas albilineans Infection under Drought Stress.

Plants must adapt to the complex effects of several stressors brought on by global warming, which may result in interaction and superposition effects between diverse stressors. Few reports are available on how drought stress affects Xanthomonas albilineans (Xa) infection in sugarcane (Saccharum spp. hybrids). Drought and leaf scald resistance were identified on 16 sugarcane cultivars using Xa inoculation and soil drought treatments, respectively. Subsequently, four cultivars contrasting to drought and leaf scald resistance were used to explore the mechanisms of drought affecting Xa-sugarcane interaction. Drought stress significantly increased the occurrence of leaf scald and Xa populations in susceptible cultivars but had no obvious effect on resistant cultivars. The ROS bursting and scavenging system was significantly activated in sugarcane in the process of Xa infection, particularly in the resistant cultivars. Compared with Xa infection alone, defense response via the ROS generating and scavenging system was obviously weakened in sugarcane (especially in susceptible cultivars) under Xa infection plus drought stress. Collectively, ROS might play a crucial role involving sugarcane defense against combined effects of Xa infection and drought stress.

Molecular, biochemical and metabolomics analyses reveal constitutive and pathogen-induced defense responses of two sugarcane contrasting genotypes against leaf scald disease

Leaf scald caused by the bacteria Xanthomonas albilineans is one of the major concerns to sugarcane production. To breed for resistance, mechanisms underlying plant-pathogen interaction need deeper investigations. Herein, we evaluated sugarcane defense responses against X. albilineans using molecular and biochemical approaches to assess pathogen-triggered ROS, phytohormones and metabolomics in two contrasting sugarcane genotypes from 0.5 to 144 h post-inoculation (hpi). In addition, the infection process was monitored using TaqMan-based quantification of X. albilineans and the disease symptoms were evaluated in both genotypes after 15 d post-inoculation (dpi). The susceptible genotype presented a response to the infection at 0.5 hpi, accumulating defense-related metabolites such as phenolics and flavonoids with no significant defense responses thereafter, resulting in typical symptoms of leaf scald at 15 dpi. The resistant genotype did not respond to the infection at 0.5 hpi but constitutively presented higher levels of salicylic acid and of the same metabolites induced by the infection in the susceptible genotype. Moreover, two subsequent pathogen-induced metabolic responses at 12 and 144 hpi were observed only in the resistant genotype in terms of amino acids, quinic acids, coumarins, polyamines, flavonoids, phenolics and phenylpropanoids together with an increase of hydrogen peroxide, ROS-related genes expression, indole-3-acetic-acid and salicylic acid. Multilevel approaches revealed that constitutive chemical composition and metabolic reprogramming hampers the development of leaf scald at 48 and 72 hpi, reducing the disease symptoms in the resistant genotype at 15 dpi. Phenylpropanoid pathway is suggested as a strong candidate marker for breeding sugarcane resistant to leaf scald.

Sugarcane responses to two strains of Xanthomonas albilineans differing in pathogenicity through a differential modulation of salicylic acid and reactive oxygen species.

Leaf scald caused by Xanthomonas albilineans is one of the major bacterial diseases of sugarcane that threaten the sugar industry worldwide. Pathogenic divergence among strains of X. albilineans and interactions with the sugarcane host remain largely unexplored. In this study, 40 strains of X. albilineans from China were distributed into three distinct evolutionary groups based on multilocus sequence analysis and simple sequence repeats loci markers. In pathogenicity assays, the 40 strains of X. albilineans from China were divided into three pathogenicity groups (low, medium, and high). Twenty-four hours post inoculation (hpi) of leaf scald susceptible variety GT58, leaf populations of X. albilineans strain XaCN51 (high pathogenicity group) determined by qPCR were 3-fold higher than those of strain XaCN24 (low pathogenicity group). Inoculated sugarcane plants modulated the reactive oxygen species (ROS) homoeostasis by enhancing respiratory burst oxidase homolog (ScRBOH) expression and superoxide dismutase (SOD) activity and by decreasing catalase (CAT) activity, especially after infection by X. albilineans XaCN51. Furthermore, at 24 hpi, plants infected with XaCN51 maintained a lower content of endogenous salicylic acid (SA) and a lower expression level of SA-mediated genes (ScNPR3, ScTGA4, ScPR1, and ScPR5) as compared to plants infected with XaCN24. Altogether, these data revealed that the ROS production-scavenging system and activation of the SA pathway were involved in the sugarcane defense response to an attack by X. albilineans.

Integrated Transcriptome and Metabolome Analysis to Identify Sugarcane Gene Defense against Fall Armyworm (Spodoptera frugiperda) Herbivory.

Sugarcane is the most important sugar crop, contributing ≥80% to total sugar production around the world. Spodoptera frugiperda is one of the main pests of sugarcane, potentially causing severe yield and sugar loss. The identification of key defense factors against S. frugiperda herbivory can provide targets for improving sugarcane resistance to insect pests by molecular breeding. In this work, we used one of the main sugarcane pests, S. frugiperda, as the tested insect to attack sugarcane. Integrated transcriptome and metabolomic analyses were performed to explore the changes in gene expression and metabolic processes that occurred in sugarcane leaf after continuous herbivory by S. frugiperda larvae for 72 h. The transcriptome analysis demonstrated that sugarcane pest herbivory enhanced several herbivory-induced responses, including carbohydrate metabolism, secondary metabolites and amino acid metabolism, plant hormone signaling transduction, pathogen responses, and transcription factors. Further metabolome analysis verified the inducement of specific metabolites of amino acids and secondary metabolites by insect herbivory. Finally, association analysis of the transcriptome and metabolome by the Pearson correlation coefficient method brought into focus the target defense genes against insect herbivory in sugarcane. These genes include amidase and lipoxygenase in amino acid metabolism, peroxidase in phenylpropanoid biosynthesis, and pathogenesis-related protein 1 in plant hormone signal transduction. A putative regulatory model was proposed to illustrate the sugarcane defense mechanism against insect attack. This work will accelerate the dissection of the mechanism underlying insect herbivory in sugarcane and provide targets for improving sugarcane variety resistance to insect herbivory by molecular breeding.

Discovery of potential protein biomarkers associated with sugarcane white leaf disease susceptibility using a comparative proteomic approach.

Sugarcane white leaf disease (SCWLD) is caused by phytoplasma, a serious sugarcane phytoplasma pathogen, which causes significant decreases in crop yield and sugar quality. The identification of proteins involved in the defense mechanism against SCWLD phytoplasma may help towards the development of varieties resistant to SCWLD. We investigated the proteomes of four sugarcane varieties with different levels of susceptibility to SCWLD phytoplasma infection, namely K88-92 and K95-84 (high), KK3 (moderate), and UT1 (low) by quantitative label-free nano-liquid chromatography-tandem mass spectrometry (nano LC-MS/MS). A total of 248 proteins were identified and compared among the four sugarcane varieties. Two potential candidate protein biomarkers for reduced susceptibility to SCWLD phytoplasma were identified as proteins detected only in UT1. The functions of these proteins are associated with protein folding, metal ion binding, and oxidoreductase. The candidate biomarkers could be useful for further study of the sugarcane defense mechanism against SCWLD phytoplasma, and in molecular and conventional breeding strategies for variety improvement.

Structural and Evolutionary Analyses of PR-4 SUGARWINs Points to a Different Pattern of Protein Function.

SUGARWINs are PR-4 proteins associated with sugarcane defense against phytopathogens. Their expression is induced in response to damage by Diatraea saccharalis larvae. These proteins play an important role in plant defense, in particular against fungal pathogens, such as Colletothricum falcatum (Went) and Fusarium verticillioides. The pathogenesis-related protein-4 (PR-4) family is a group of proteins equipped with a BARWIN domain, which may be associated with a chitin-binding domain also known as the hevein-like domain. Several PR-4 proteins exhibit both chitinase and RNase activity, with the latter being associated with the presence of two histidine residues H11 and H113 (BARWIN) [H44 and H146, SUGARWINs] in the BARWIN-like domain. In sugarcane, similar to other PR-4 proteins, SUGARWIN1 exhibits ribonuclease, chitosanase and chitinase activities, whereas SUGARWIN2 only exhibits chitosanase activity. In order to decipher the structural determinants involved in this diverse range of enzyme specificities, we determined the 3-D structure of SUGARWIN2, at 1.55Å by X-ray diffraction. This is the first structure of a PR-4 protein where the first histidine has been replaced by asparagine and was subsequently used to build a homology model for SUGARWIN1. Molecular dynamics simulations of both proteins revealed the presence of a flexible loop only in SUGARWIN1 and we postulate that this, together with the presence of the catalytic histidine at position 42, renders it competent as a ribonuclease. The more electropositive surface potential of SUGARWIN1 would also be expected to favor complex formation with RNA. A phylogenetic analysis of PR-4 proteins obtained from 106 Embryophyta genomes showed that both catalytic histidines are widespread among them with few replacements in these amino acid positions during the gene family evolutionary history. We observe that the H11 replacement by N11 is also present in two other sugarcane PR-4 proteins: SUGARWIN3 and SUGARWIN4. We propose that RNase activity was present in the first Embryophyta PR-4 proteins but was recently lost in members of this family during the course of evolution.

Unveiling sugarcane defense response to Mythimna separata herbivory by a combination of transcriptome and metabolic analyses.

Sugarcane is the most important sugar crop in the world. Like other crops, sugarcane suffers from herbivorous insect attack. The oriental armyworm Mythimna separata is a devastating pest of various crops in northeast Asia and an outbreak of this pest can result in substantial yield loss for sugarcane. However, the plant defense response situation is widely acquisition in model crops, but there is little information about how sugarcane plants defend themselves against this herbivore at the molecular and biochemical levels. We combined transcriptome and metabolomic analysis to investigate the changes in gene expression and metabolic processes that occurred in sugarcane plants after continuous feeding by M. separata larvae for 12 and 24 h. We identified 13 662 genes and 55 metabolites that were differentially regulated in sugarcane plants fed on by M. separata. The genes involved in phytohormones, transcription factors, and kinase-related were activated and metabolism compounds such as carbohydrate, amino acid, ferulic substances and glutathione were detected regulated in sugarcane defense response. Comparable analyses showed a close correspondence relationship among pathways of phenylalanine metabolism, phenylpropanoid biosynthesis, and flavonoid biosynthesis in transcript and metabolite profiles. Furthermore, a bioassay experiment was conducted to test the influence of up-regulated metabolites on M. separata growth and found chlorogenic acid had a lethal effect. The results of our study greatly enhanced the understanding of the sugarcane-induced defense response mechanism against herbivore infestation at the transcriptional and metabolic levels. Also make contributions to provide clues for development of green pest control method. © 2021 Society of Chemical Industry.

Role of miRNAs in the host-pathogen interaction between sugarcane and Colletotrichum falcatum, the red rot pathogen.

Sugarcane microRNAs specifically involved during compatible and incompatible interactions with red rot pathogen Colletotrichum falcatum were identified. We have identified how the miRNAs regulate their gene targets and elaborated evidently on the underlying molecular mechanism of sugarcane defense response to C. falcatum for the first time. Resistance against the fungal pathogen Colletotrichum falcatum causing red rot is one of the most desirable traits for sustainable crop cultivation in sugarcane. To gain new insight into the host defense mechanism against C. falcatum, we studied the role of sugarcane microRNAs during compatible and incompatible interactions by adopting the NGS platform. We have sequenced a total of 80 miRNA families that comprised 980 miRNAs, and the putative targets of the miRNAs include transcription factors, membrane-bound proteins, glutamate receptor proteins, lignin biosynthesis proteins, signaling cascade proteins, transporter proteins, mitochondrial proteins, ER proteins, defense-related, stress response proteins, translational regulation proteins, cell proliferation, and ubiquitination proteins. Further, qRT-PCR analyses of 8 differentially regulated miRNAs and 26 gene transcript targets expression indicated that these miRNAs have a regulatory effect on the expression of respective target genes in most of the cases. Also, the results suggest that certain miRNA regulates many target genes that are involved in inciting early responses to the pathogen infection, signaling pathways, endoplasmic reticulum stress, and resistance gene activation through feedback response from various cellular processes during the compatible and incompatible interaction with the red rot pathogen C. falcatum. The present study revealed the role of sugarcane miRNAs and their target genes during sugarcane-C. falcatum interaction and provided new insight into the miRNA-mediated defense mechanism in sugarcane for the first time.

Quantitative proteomic analysis of the sugarcane defense responses incited by Acidovorax avenae subsp. avenae causing red stripe

The causal agent of red stripe, Acidovorax avenae subsp. avenae (Aaa), can incite significant economic losses during sugarcane production. Proteomics is a useful tool for the comprehensive identification of defense response proteins in plants. In this study, isobaric tags for relative and absolute quantification (iTRAQ)-based proteomics was used to identify proteins that were produced in sugarcane in defense to Aaa in two Saccharum spp. cultivars: ROC22 (resistant to red stripe) and MT11−610 (susceptible). Defense response was evaluated at 0 h (mock-inoculation) and 24 h after inoculation with the bacterial pathogen, and 10,502 high-confidence proteins and 1962 differentially expressed proteins (DEPs) were identified. These DEPs included 1027 proteins (671 upregulated, 354 downregulated) in ROC22 and 1130 proteins (566 upregulated, 564 downregulated) in MT11−610. Gene ontology analysis revealed that most DEPs were clustered in the metabolic, single-organism metabolic, and oxidation-reduction processes, and in catalytic activity. In a KEGG analysis, these proteins belonged to common pathways such as secondary metabolite biosynthesis and biosynthesis of phenylpropanoids. Thirty-six DEPs were identified, especially epoxide hydrolase 1, dynamin-related protein 1C-like, cytochrome oxidase subunit I, FK506-binding protein, acetoacetyl CoA thiolase isoform 2, anthocyanidin 3-o-glucosyltransferase, ubiquitin carboxyl-terminal hydrolase 4, protein disulfide isomerase-like protein, and jumonji-like transcription factor family protein. These nine proteins were characterized by enhanced expression with ≥1.5-fold increases in resistant ROC22 versus susceptible MT11−610, and are therefore biomarker candidates in the sugarcane response against Aaa. These data could be very useful in molecular breeding for sugarcane resistance to red stripe.

Selection of reference genes for normalization of microRNA expression in sugarcane stalks during its interaction with Colletotrichum falcatum.

The microRNAs role in various cellular and metabolic functions is gaining more limelight in line with second-generation NGS technology. For the validation of candidate miRNA genes, the quantitative real-time PCR is the widely trusted and efficient method to follow. Sugarcane miRNAs are less explored in sugarcane defense response during their interaction with Colletotrichum falcatum inciting red rot. Further, for RT-qPCR experiments involving sugarcane miRNA expression studies, a stable internal reference gene is required. Hence, we have taken a study involving 20 candidate genes to identify stable expressing reference genes using NormFinder, geNorm, BestKeeper, and deltaCt statistical algorithms. The candidate reference genes included miRNAs and protein-coding genes. The results indicated that there is a variation in ranking among the algorithms. We found miR1862c as the stably expressed miRNA reference gene among the candidates and miR444b.2 along miR1862c formed the best reference gene pair combination, which can be used in the experiments aiming to explore sugarcane miRNAs in the defense mechanism against C. falcatum. The stable miRNA reference gene was further validated with other lesser stable reference gene candidates to assess the effect of stable reference genes during normalization. The present study evaluating the sugarcane miRNAs as reference genes for normalizing RT-qPCR expression data involving miRNAs during sugarcane ×C. falcatum interaction is the first of its kind. Further, this systematic approach can be followed to assess the reference gene in various experimental conditions involving sugarcane miRNAs.

Metabolic and proteomic analysis of nitrogen metabolism mechanisms involved in the sugarcane – Fusarium verticillioides interaction

Pokkah boeng disease (PBD) is a foliar disease causing severe losses in sugarcane crop production. Research into resistance mechanisms against the causal agent, Fusarium verticillioides, is particularly important for farmers and researchers. This work based on the comprehensive analysis of metabolic, proteomic, and bioinformatics data on nitrogen (N) metabolism, which revealed that this biosynthetic reactions was closely related to resistance mechanisms in the sugarcane– F. verticillioides interaction. Our results suggested that pathogen infection reduced the suppression of nitrate reductase (NR) activity, reducing ammonium nitrogen (NH4+-N) and nitrate nitrogen (NO3−-N) assimilation, which reduces glutamine synthetase (GS), glutamate synthetase (GOGAT) activity and polynucleotide synthesis and promotes RNA degradation, resulting in a decrease in ribosome levels and protein synthesis. Cysteine was found to be associated with the symptoms of PBD, while alanine, lysine, proline, and glutamic acid were found to be involved in protective and regulatory mechanisms as well. Additionally, glutamate played an important role in sugarcane defense against pathogens through the biosynthesis of proline and polyamines. Cyanamide, glutamate, proline, tyrosine, and arachidonic acid metabolism actively participate in resistance and response to stress. C5XPZ6 and C5XCA6 were considered to be critical proteins and key effectors according to this study. In conclusion, we have identified potential proteins and pathways involved in sugarcane resistance to F. verticillioides, revealing new findings that may be useful in the design of future diagnostics or sugarcane protection strategies and providing new insights into the molecular mechanisms of sugarcane–pathogen interactions.

Screening of environmental stress tolerant superior sugarcane (Saccharum officinarum L.)

Sugarcane (Saccharum officinarum L.) is among the important commodities of the commercial sector since it accounts for 65% of the world’s sugar production. Indonesia is the world’s eighth largest country in terms of productivity, planting area and yield of sugarcane. Environmental stresses due to climate change are among factors that may cause negative impacts on the production of sugarcane. The purpose of the present study was to determine sugarcane cultivars tolerant to environmental stress based on a multiplex analysis of SCDR 1 and P5CS, as well as sugarcane defense mechanisms against environmental stress conditions. The methods used in this study were quantitative test with spectrophotometry, qualitative test with electrophoresis, DNA amplification of the target SCDR 1 and P5CS by the PCR method. The presence of these two genes in the tested sugarcane cultivars indicates a tolerant trait of environmental stress. The study found that 24 tested sugarcane cultivars are potentially tolerant to environmental stress due to the presence of those two genes.

Global Gene Responses of Resistant and Susceptible Sugarcane Cultivars to Acidovorax avenae subsp. avenae Identified Using Comparative Transcriptome Analysis.

Red stripe disease in sugarcane caused by Acidovorax avenae subsp. avenae (Aaa) is related to serious global losses in yield. However, the underlying molecular mechanisms associated with responses of sugarcane plants to infection by this pathogen remain largely unknown. Here, we used Illumina RNA-sequencing (RNA-seq) to perform large-scale transcriptome sequencing of two sugarcane cultivars to contrast gene expression patterns of plants between Aaa and mock inoculations, and identify key genes and pathways involved in sugarcane defense responses to Aaa infection. At 0–72 hours post-inoculation (hpi) of the red stripe disease-resistant cultivar ROC22, a total of 18,689 genes were differentially expressed between Aaa-inoculated and mock-inoculated samples. Of these, 8498 and 10,196 genes were up- and downregulated, respectively. In MT11-610, which is susceptible to red stripe disease, 15,782 genes were differentially expressed between Aaa-inoculated and mock-inoculated samples and 8807 and 6984 genes were up- and downregulated, respectively. The genes that were differentially expressed following Aaa inoculation were mainly involved in photosynthesis and carbon metabolism, phenylpropanoid biosynthesis, plant hormone signal transduction, and plant–pathogen interaction pathways. Further, qRT-PCR and RNA-seq used for additional validation of 12 differentially expressed genes (DEGs) showed that eight genes in particular were highly expressed in ROC22. These eight genes participated in the biosynthesis of lignin and coumarin, as well as signal transduction by salicylic acid, jasmonic acid, ethylene, and mitogen-activated protein kinase (MAPK), suggesting that they play essential roles in sugarcane resistance to Aaa. Collectively, our results characterized the sugarcane transcriptome during early infection with Aaa, thereby providing insights into the molecular mechanisms responsible for bacterial tolerance.

Transcriptional analysis identifies major pathways as response components to Sporisorium scitamineum stress in sugarcane

BackgroundSugarcane smut, which is caused by Sporisorium scitamineum, is a severe fungal disease affecting sugarcane. However, the major pathways involved in the interaction between sugarcane and S. scitamineum remains unclear. ResultsIn the present study, suppression subtractive hybridization (SSH) library construction, together with reverse northern blotting, was conducted on the most prevalent sugarcane genotype ROC22 challenged with S. scitamineum. After alignment and homologous expressed sequence tag (EST) assembly, a total of 155 differentially expressed unigenes were identified from SSH libraries. Totally, 26 of 155 differentially expressed unigenes were analyzed by qRT-PCR in sugarcane smut-resistant genotype YC05-179 and susceptible genotype ROC22. Genes encoded two unknown protein (Q1 and Q11), serine/threonine kinase (Q2), fiber protein (Q3), eukaryotic translation initiation factor 5A (Q23), and Sc14-3-3-like protein (Q24) were induced in sugarcane smut-resistant genotype YC05-179 but inhibited in susceptible genotype ROC22. Based on the differential expression data achieved from SSH libraries and qRT-PCR, we found that, serine/threonine kinases, Ca2+ sensors, mitogen-activated protein genes and some NBS-LRR genes may involve in the signal recognition and transduction of smut fungus infection in sugarcane. While in the plant hormone signaling pathways, the genes related to auxin, abscisic acid, salicylic acid and ethylene were more apparently in response to smut fungus invasion. The hypersensitive response, protein metabolism, polyamine synthesis, and cell wall formation may play an important role in sugarcane defense against smut fungus colonization. Additionally, the Sc14-3-3 might serve as a molecular modulator in sugarcane being immune to smut disease by interacting with proteins like ScGAPN (Q10), which have been further verified by BiFC assay. ConclusionsThe findings of the present study could provide a general view about gene pathways involving in sugarcane defense against smut disease and facilitate a better understanding of the molecular mechanism underlying sugarcane-S. scitamineum interaction.

A Small GTP-Binding Gene ScRan from Sugarcane is Involved in Responses to Various Hormone Stresses and Sporisirium scitamineum Challenge

The ras-like nuclear GTP-binding protein (Ran) is known as a molecular switch and plays an important role in eukaryotic metabolism. In this study, a differentially expressed EST sequence from our previous suppression subtractive hybridization libraries of sugarcane under Sporisirium scitamineum infection was used as the probe, and an 836-bp-long sugarcane Ran gene (ScRan) was cloned from sugarcane smut-resistant genotype YC05-179. Bioinformatic analysis revealed that the ScRan gene contained a 666-bp-long complete open-reading frame, encoding a stable 25-kDa acidic protein with four core domains GDGGTGKT (I), DTAG (II), NKVD (III), and EISAK (IV), and without the signal peptide. The inducing expression results of ScRan in Escherichia coli showed that the molecular weight of ScRan was about 20–35 kDa. Sequence alignment and phylogenetic analysis indicated that ScRan protein was conservative in evolution. ScRan protein was located in both the nucleus and the cytoplasm and could interact with sugarcane Sc14-3-3 protein (GenBank Acc. No.: AY222859). qRT-PCR analysis demonstrated that the expression of ScRan was higher in root than all the other sugarcane tissues including bud, leaf sheath, stem pith, and epidermis. The upregulation of ScRan in case of abscisic acid (ABA), salicylic acid (SA), and methyl jasmonic acid (MeJA) stimuli indicated its positive involvement in sugarcane responses to phytohormone ABA, SA, and MeJA signaling. ScRan was positively responded to S. scitamineum infection in the early stage but was inhibited in the later stage. These results would facilitate understanding the important role of the ScRan gene in sugarcane defense against various hormone stresses and S. scitamineum stress.

A sugarcane pathogenesis-related protein, ScPR10, plays a positive role in defense responses under Sporisorium scitamineum, SrMV, SA, and MeJA stresses.

A pathogenesis-related gene, ScPR10 , was isolated from sugarcane and its bio-function was characterized, demonstrating that ScPR10 was involved in plant defense responses to Sporisorium scitamineum , SrMV, SA, and MeJA stresses. Plant fungal and viral diseases are the major concerns in sugarcane industry. Many anti-fungal and antivirus components, including pathogenesis-related (PR) proteins, have been identified. The pathogenesis-related protein 10 (PR10) is the dominant group in PR families, involved in the plant defense mechanism. In this study, ScPR10 (GenBank Acc. No. KT887884), a 701-bp-length PR10 gene with a 483bp-length open reading frame, was isolated from sugarcane. Its transient expression in the leaves of Nicotiana benthamiana indicated that the function role of ScPR10 is likely in the nucleus, and it increased the level of H2O2 accumulation in leaf cells. Moreover, ScPR10 could also enhance the resistance of N. benthamiana leaves to infection by Pseudomonas solanacearum and Fusarium solani var. coeruleum. Quantitative real-time PCR analysis revealed that ScPR10 was not constitutively expressed in sugarcane tissues due to its high expression in the buds and scant presence in root tips. In addition, the transcript of ScPR10 could be induced by a pathogenic fungus (Sporisorium scitamineum) and a virus (Sorghum mosaic virus, SrMV) in the resistant sugarcane cultivars, while it was down-regulated in the susceptible ones. After exposure to salicylic acid (SA) and methyl jasmonate (MeJA), ScPR10 peaked at 6 and 12h, respectively. These results suggest that ScPR10 can play a positive role in sugarcane defense responses to S. scitamineum, SrMV, SA, and MeJA stresses.

The sugarcane defense protein SUGARWIN2 causes cell death in Colletotrichum falcatum but not in non-pathogenic fungi.

Plants respond to pathogens and insect attacks by inducing and accumulating a large set of defense-related proteins. Two homologues of a barley wound-inducible protein (BARWIN) have been characterized in sugarcane, SUGARWIN1 and SUGARWIN2 (sugarcane wound-inducible proteins). Induction of SUGARWINs occurs in response to Diatraea saccharalis damage but not to pathogen infection. In addition, the protein itself does not show any effect on insect development; instead, it has antimicrobial activities toward Fusarium verticillioides, an opportunistic fungus that usually occurs after D. saccharalis borer attacks on sugarcane. In this study, we sought to evaluate the specificity of SUGARWIN2 to better understand its mechanism of action against phytopathogens and the associations between fungi and insects that affect plants. We used Colletotrichum falcatum, a fungus that causes red rot disease in sugarcane fields infested by D. saccharalis, and Ceratocystis paradoxa, which causes pineapple disease in sugarcane. We also tested whether SUGARWIN2 is able to cause cell death in Aspergillus nidulans, a fungus that does not infect sugarcane, and in the model yeast Saccharomyces cerevisiae, which is used for bioethanol production. Recombinant SUGARWIN2 altered C. falcatum morphology by increasing vacuolization, points of fractures and a leak of intracellular material, leading to germling apoptosis. In C. paradoxa, SUGARWIN2 showed increased vacuolization in hyphae but did not kill the fungi. Neither the non-pathogenic fungus A. nidulans nor the yeast S. cerevisiae was affected by recombinant SUGARWIN2, suggesting that the protein is specific to sugarcane opportunistic fungal pathogens.

Molecular cloning and characterization of two pathogenesis-related β-1,3-glucanase genes ScGluA1 and ScGluD1 from sugarcane infected by Sporisorium scitamineum

Two β-1,3-glucanase genes from sugarcane were cloned and characterized. They were all located in apoplast and involves in different expression patterns in biotic and abiotic stress. Smut caused by Sporisorium scitamineum is a serious disease in the sugarcane industry. β-1,3-Glucanase, a typical pathogenesis-related protein, has been shown to express during plant-pathogen interaction and involves in sugarcane defense response. In this study, β-1,3-glucanase enzyme activity in the resistant variety increased faster and lasted longer than that of the susceptible one when inoculated with S. scitamineum, along with a positive correlation between the activity of the β-1,3-glucanase and smut resistance. Furthermore, two β-1,3-glucanase genes from S. scitamineum infected sugarcane, ScGluA1 (GenBank Accession No. KC848050) and ScGluD1 (GenBank Accession No. KC848051) were cloned and characterized. Phylogenetic analysis suggested that ScGluA1 and ScGluD1 clustered within subfamily A and subfamily D, respectively. Subcellular localization analysis demonstrated that both gene products were targeted to apoplast. Escherichia coli Rosetta (DE3) cells expressing ScGluA1 and ScGluD1 showed varying degrees of tolerance to NaCl, CdCl2, PEG, CuCl2 and ZnSO4. Q-PCR analysis showed up-regulation of ScGluA1 and slight down-regulation of ScGluD1 in response to S. scitamineum infection. It suggested that ScGluA1 may be involved in the defense reaction of the sugarcane to the smut, while it is likely that ScGluD1 was inhibited. The gene expression patterns of ScGluA1 and ScGluD1, in response to abiotic stresses, were similar to sugarcane response against smut infection. Together, β-1,3-glucanase may function in sugarcane defense mechanism for S. scitamineum. The positive responses of ScGluA1 and the negative responses of ScGluD1 to biotic and abiotic stresses indicate they play different roles in interaction between sugarcane and biotic or abiotic stresses.

Transcriptome profile analysis of sugarcane responses to Sporisorium scitaminea infection using Solexa sequencing technology.

To understand the molecular basis of sugarcane-smut interaction, it is important to identify sugarcane genes that respond to the pathogen attack. High-throughput tag-sequencing (tag-seq) analysis by Solexa technology was performed on sugarcane infected with Sporisorium scitaminea, which should have massively increased the amount of data available for transcriptome profile analysis. After mapping to sugarcane EST databases in NCBI, we obtained 2015 differentially expressed genes, of which 1125 were upregulated and 890 downregulated by infection. Gene ontology (GO) analysis revealed that the differentially expressed genes involve in many cellular processes. Pathway analysis revealed that metabolic pathways and ribosome function are significantly affected, where upregulation of expression dominates over downregulation. Differential expression of three candidate genes involved in MAP kinase signaling pathway, ScBAK1 (GenBank Accession number: KC857629), ScMapkk (GenBank Accession number: KC857627), and ScGloI (GenBank Accession number: KC857628), was confirmed by reverse transcription polymerase chain reaction (RT-PCR). Real-time quantitative PCR (qRT-PCR) analysis concluded that the expression of these genes were all up-regulated after the infection of S. scitaminea and may play a role in pathogen response in sugarcane. The present study provides insights into the molecular mechanism of sugarcane defense to S. scitaminea infection, leading to a more comprehensive understanding of sugarcane-smut interaction.