Sugarcane ScOPR1 gene enhances plant disease resistance through the modulation of hormonal signaling pathways.

KEY MESSAGE: Transgenic plants stably overexpressing ScOPR1 gene enhanced disease resistance by increasing the accumulation of JA, SA, and GST, as well as up-regulating the expression of genes related to signaling pathways. 12-Oxo-phytodienoate reductase (OPR) is an oxidoreductase that depends on flavin mononucleotide (FMN) and catalyzes the conversion of 12-oxophytodienoate (12-OPDA) into jasmonic acid (JA). It plays a key role in plant growth and development, and resistance to adverse stresses. In our previous study, we have obtained an OPR gene (ScOPR1, GenBank Accession Number: MG755745) from sugarcane. This gene showed positive responses to methyl jasmonate (MeJA), salicylic acid (SA), abscisic acid (ABA), and Sporisorium scitamineum, suggesting its potential for pathogen resistance. Here, in our study, we observed that Nicotiana benthamiana leaves transiently overexpressing ScOPR1 exhibited weaker disease symptoms, darker 3,3-diaminobenzidine (DAB) staining, higher accumulation of reactive oxygen species (ROS), and higher expression of hypersensitive response (HR) and SA pathway-related genes after inoculation with Ralstonia solanacearum and Fusarium solanacearum var. coeruleum. Furthermore, the transgenic N. benthamiana plants stably overexpressing the ScOPR1 gene showed enhanced resistance to pathogen infection by increasing the accumulation of JA, SA, and glutathione S-transferase (GST), as well as up-regulating genes related to HR, JA, SA, and ROS signaling pathways. Transcriptome analysis revealed that the specific differentially expressed genes (DEGs) in ScOPR1-OE were significantly enriched in hormone transduction signaling and plant-pathogen interaction pathways. Finally, a functional mechanism model of the ScOPR1 gene in response to pathogen infection was depicted. This study provides insights into the molecular mechanism of ScOPR1 and presents compelling evidence supporting its positive involvement in enhancing plant disease resistance.

Genome-wide characterization of sugarcane catalase gene family identifies a ScCAT1 gene associated disease resistance.

In plants, catalase (CAT) mainly scavenges H2O2 from reactive oxygen species (ROS) and regulates the growth and development. So far, genome-wide identification of CAT gene family in Saccharum has not yet been reported. Here, 16 SsCAT genes were identified based on a Saccharum spontaneum genome. They were clustered into three subfamilies, with closer genes sharing similar structures. Most SsCAT proteins contained three conserved amino acids, one active catalytic site, one heme-ligand signature, and three peroxisomal targeting signal 1 (PTS1) sequences. The cis-regulatory element prediction revealed that SsCAT genes were involved in growth and development, and in response to various hormones and stresses. RNA-Seq databases showed that SsCAT genes were differentially expressed in Saccharum tissues and under cold, drought, and Sporisorium scitamineum stresses. The ScCAT1 gene transcript (GenBank accession number KF664183) and relevant CAT activity were up-regulated under S. scitamineum stress. Overexpression of ScCAT1 gene in Nicotiana benthamiana could enhance its resistance to pathogen infection through scavenging of excessive toxic ROS and up-regulated expressions of genes related to hypersensitive response (HR), ROS and salicylic acid (SA) pathways. This study provides comprehensive information for the CAT gene family and sets up a basis for its function identification in sugarcane.

Identification and characterization of WAK gene family in Saccharum and the negative roles of ScWAK1 under the pathogen stress.

Wall-associated kinase (WAK) is widely involved in signal transduction, reproductive growth, responses to pathogen infection and metal ion stress in plants. In this study, 19, 12, and 37 SsWAK genes were identified in Saccharum spontaneum, Saccharum hybrid and Sorghum bicolor, respectively. Phylogenetic tree showed that they could be divided into three groups. These WAK genes contained multiple cis-acting elements related to stress, growth and hormone response. RNA-seq analysis demonstrated that SsWAK genes were constitutively expressed in different sugarcane tissues and involved in response to smut pathogen (Sporisorium scitamineum) stress. Additionally, ScWAK1 (GenBank Accession No. OP479864), was then isolated from sugarcane cultivar ROC22. It was highly expressed in leaves and roots and its expression could be induced under SA and MeJA stress. Besides, ScWAK1 was significantly downregulated in both smut-resistant and susceptible sugarcane cultivars in response to S. scitamineum infection. ScWAK1 was a membrane protein without self-activating activity. Furthermore, transient expression of ScWAK1 in Nicotiana benthamiana enhanced the susceptibility of tobacco to the inoculation of Ralstonia solanacearum and Fusarium solani var. coeruleum, suggesting its negative role in disease resistance. The present study reveals the origin, distribution and evolution of WAK gene family and provides potential gene resources for sugarcane molecular breeding.

Genome-Wide Identification of Auxin-Responsive GH3 Gene Family in Saccharum and the Expression of ScGH3-1 in Stress Response.

Gretchen Hagen3 (GH3), one of the three major auxin-responsive gene families, is involved in hormone homeostasis in vivo by amino acid splicing with the free forms of salicylic acid (SA), jasmonic acid (JA) or indole-3-acetic acid (IAA). Until now, the functions of sugarcane GH3 (SsGH3) family genes in response to biotic stresses have been largely unknown. In this study, we performed a systematic identification of the SsGH3 gene family at the genome level and identified 41 members on 19 chromosomes in the wild sugarcane species, Saccharum spontaneum. Many of these genes were segmentally duplicated and polyploidization was the main contributor to the increased number of SsGH3 members. SsGH3 proteins can be divided into three major categories (SsGH3-I, SsGH3-II, and SsGH3-III) and most SsGH3 genes have relatively conserved exon-intron arrangements and motif compositions. Diverse cis-elements in the promoters of SsGH3 genes were predicted to be essential players in regulating SsGH3 expression patterns. Multiple transcriptome datasets demonstrated that many SsGH3 genes were responsive to biotic and abiotic stresses and possibly had important functions in the stress response. RNA sequencing and RT-qPCR analysis revealed that SsGH3 genes were differentially expressed in sugarcane tissues and under Sporisorium scitamineum stress. In addition, the SsGH3 homolog ScGH3-1 gene (GenBank accession number: OP429459) was cloned from the sugarcane cultivar (Saccharum hybrid) ROC22 and verified to encode a nuclear- and membrane-localization protein. ScGH3-1 was constitutively expressed in all tissues of sugarcane and the highest amount was observed in the stem pith. Interestingly, it was down-regulated after smut pathogen infection but up-regulated after MeJA and SA treatments. Furthermore, transiently overexpressed Nicotiana benthamiana, transduced with the ScGH3-1 gene, showed negative regulation in response to the infection of Ralstonia solanacearum and Fusarium solani var. coeruleum. Finally, a potential model for ScGH3-1-mediated regulation of resistance to pathogen infection in transgenic N. benthamiana plants was proposed. This study lays the foundation for a comprehensive understanding of the sequence characteristics, structural properties, evolutionary relationships, and expression of the GH3 gene family and thus provides a potential genetic resource for sugarcane disease-resistance breeding.

Genetic identification of SNP markers and candidate genes associated with sugarcane smut resistance using BSR-Seq.

Sugarcane smut caused by Sporisorium scitamineum is one of the most severe fungal diseases worldwide. In this study, a cross was made between a smut-resistant variety YT93-159 and a smut-susceptible variety ROC22, and 312 progenies were obtained. Two bulks of progenies were then constructed, one consisted of 27 highly smut resistant progenies and the other 24 smut susceptible progenies. Total RNAs of the progenies of each bulk, were pooled and subject to bulked segregant RNA-sequence analysis (BSR-Seq). A total of 164.44 Gb clean data containing 2,341,449 SNPs and 64,999 genes were obtained, 7,295 of which were differentially expressed genes (DEGs). These DEGs were mainly enriched in stress-related metabolic pathways, including carbon metabolism, phenylalanine metabolism, plant hormone signal transduction, glutathione metabolism, and plant-pathogen interactions. Besides, 45,946 high-quality, credible SNPs, a 1.27 Mb region at Saccharum spontaneum chromosome Chr5B (68,904,827 to 70,172,982), and 129 candidate genes were identified to be associated with smut resistance. Among them, twenty-four genes, either encoding key enzymes involved in signaling pathways or being transcription factors, were found to be very closely associated with stress resistance. RT-qPCR analysis demonstrated that they played a positive role in smut resistance. Finally, a potential molecular mechanism of sugarcane and S. scitamineum interaction is depicted that activations of MAPK cascade signaling, ROS signaling, Ca2+ signaling, and PAL metabolic pathway and initiation of the glyoxalase system jointly promote the resistance to S. scitamineum in sugarcane. This study provides potential SNP markers and candidate gene resources for smut resistance breeding in sugarcane.