A Cotyledon-based Virus-Induced Gene Silencing (Cotyledon-VIGS) approach to study specialized metabolism in medicinal plants.

BACKGROUND: Virus-induced gene silencing (VIGS) is widely used in plant functional genomics. However, the efficiency of VIGS in young plantlets varies across plant species. Additionally, VIGS is not optimized for many plant species, especially medicinal plants that produce valuable specialized metabolites. RESULTS: We evaluated the efficacy of five-day-old, etiolated seedlings of Catharanthus roseus (periwinkle) for VIGS. The seedlings were vacuum-infiltrated with Agrobacterium tumefaciens GV3101 cells carrying the tobacco rattle virus (TRV) vectors. The protoporphyrin IX magnesium chelatase subunit H (ChlH) gene, a key gene in chlorophyll biosynthesis, was used as the target for VIGS, and we observed yellow cotyledons 6 days after infiltration. As expected, the expression of CrChlH and the chlorophyll contents of the cotyledons were significantly decreased after VIGS. To validate the cotyledon based-VIGS method, we silenced the genes encoding several transcriptional regulators of the terpenoid indole alkaloid (TIA) biosynthesis in C. roseus, including two activators (CrGATA1 and CrMYC2) and two repressors (CrGBF1 and CrGBF2). Silencing CrGATA1 led to downregulation of the vindoline pathway genes (T3O, T3R, and DAT) and decreased vindoline contents in cotyledons. Silencing CrMYC2, followed by elicitation with methyl jasmonate (MeJA), resulted in the downregulation of ORCA2 and ORCA3. We also co-infiltrated C. roseus seedlings with TRV vectors that silence both CrGBF1 and CrGBF2 and overexpress CrMYC2, aiming to simultaneous silencing two repressors while overexpressing an activator. The simultaneous manipulation of repressors and activator resulted in significant upregulation of the TIA pathway genes. To demonstrate the broad application of the cotyledon-based VIGS method, we optimized the method for two other valuable medicinal plants, Glycyrrhiza inflata (licorice) and Artemisia annua (sweet wormwood). When TRV vectors carrying the fragments of the ChlH genes were infiltrated into the seedlings of these plants, we observed yellow cotyledons with decreased chlorophyll contents. CONCLUSIONS: The widely applicable cotyledon-based VIGS method is faster, more efficient, and easily accessible to additional treatments than the traditional VIGS method. It can be combined with transient gene overexpression to achieve simultaneous up- and down-regulation of desired genes in non-model plants. This method provides a powerful tool for functional genomics of medicinal plants, facilitating the discovery and production of valuable therapeutic compounds.

Identification and Characterization of Transcription Factors Regulating Terpenoid Indole Alkaloid Biosynthesis in Catharanthus roseus.

Biosynthesis of the therapeutically valuable terpenoid indole alkaloids (TIAs), in the medicinal plant Catharanthus roseus, is one of the most elaborate and complex metabolic processes. Although genomic and transcriptomic resources have significantly accelerated gene discovery in the TIA pathway, relatively few genes of transcription factors (TFs) have been identified and characterized thus far. Systematic identification of TFs and elucidation of their functions are crucial for understanding TIA pathway regulation. The successful discovery of TFs in the TIA pathway has relied mostly on three different approaches, (1) identification of cis-regulatory motifs (CRMs) present in the pathway gene promoters as they often provide clues on potential TFs that bind to the promoters, (2) co-expression analysis, based on the assumption that TFs regulating a metabolic or developmental pathway exhibit similar spatiotemporal expression as the pathway genes, and (3) isolation of homologs of TFs known to regulate structurally similar or diverse specialized metabolites in different plant species. TFs regulating TIA pathway have been isolated using either an individual or a combination of the three approaches. Here we describe transcriptome-based coexpression analysis and cis-element determination to identify TFs in C. roseus. In addition, we describe the protocols for generation of transgenic hairy roots, Agrobacterium infiltration of flowers, and electrophoretic mobility shift assay (EMSA). The methods described here are useful for the identification and characterization of potential TFs involved in the regulation of special metabolism in other medicinal plants.

Virus-Induced Gene Silencing as a Tool to Study Regulation of Alkaloid Biosynthesis in Medicinal Plants.

Advancements in genomics and transcriptomics have generated invaluable resources for the discovery of novel genes related to complex specialized metabolic pathways in plants. Virus-induced gene silencing (VIGS) has emerged as a powerful tool that is widely used for rapid functional characterization of genes in planta. VIGS has advantages over other reverse genetic approaches, such as RNAi-mediated suppression or T-DNA knockout, because it does not require the development of stable transgenic lines which is technically challenging and time consuming. Catharanthus roseus is an important medicinal plant that produces more than a hundred monoterpenoid indole alkaloids (MIAs), including the antineoplastic drugs vincristine and vinblastine. Biosynthesis of these alkaloids is strikingly complex, resulting in MIA accumulation in low quantities. Jasmonic acid (JA) is an elicitor of the MIA biosynthesis. Exogenous application of JA in C. roseus induces MIA pathway gene expression and increases MIA accumulation. The core JA signaling module comprises multiple components including the JA coreceptor Coronatine-Insensitive 1(COI1). COI1 plays a key role in JA-responsive gene expression in plants. Because generation of stable transgenic C. roseus plants is challenging, VIGS is being used for functional characterization of genes in the MIA pathway. Here we describe a detailed method for the VIGS-mediated suppression of C. roseus COI1(CrCOI1) expression to decipher the regulatory mechanism of JA-induced elicitation of MIA biosynthesis. When performing VIGS, gene silencing efficiency and the viral spread are monitored by the development of visible phenotype in the control plants. We use the C. roseus phytoene desaturase (CrPDS) and Protoporphyrin IX Mg-chelatase subunit H (CrChlH) as visual markers to access VIGS efficiency and viral spread. The protocol described here could be used for the functional characterization of genes involved in other metabolic pathways and in other medicinal plants.

Alien introgression and morpho-agronomic characterization of diploid progenies of Solanum lycopersicoides monosomic alien addition lines (MAALs) toward pre-breeding applications in tomato (S. lycopersicum).

KEY MESSAGE: Alien introgressions that were captured in the genome of diploid plants segregating from progenies of monosomic alien addition lines of S. lycopersicoides confer novel phenotypes with commercial and agronomic value in tomato breeding. Solanum lycopersicoides is a wild relative of tomato with a natural adaptation to a wide array of biotic and abiotic challenges. In this study, we identified and characterized diploid plants segregating from the progenies of monosomic alien addition lines (MAALs) of S. lycopersicoides to establish their potential as donors in breeding for target trait improvement in tomato. Molecular genotyping identified 28 of 38 MAAL progenies having the complete chromosome complement of the cultivated tomato parent and limited chromosome introgressions from the wild S. lycopersicoides parent. Analysis of SSR and indel marker profiles identified 34 unique alien introgressions in the 28 MAAL-derived introgression lines (MDILs) in the genetic background of tomato. Conserved patterns of alien introgressions were detected among sibs of MDILs 2, 3, 4 and 8. Across MDILs, a degree of preferential transmission of specific chromosome segments was also observed. Morphologically, the MDILs closely resembled the cultivated tomato more than S. lycopersicoides. The appearance of novel phenotypes in the MDILs that are lacking in the cultivated parent or the source MAALs indicates the capture of novel genetic variation by the diploid introgression lines that can add commercial and agronomic value to tomato. In particular, screening of representative MDILs for drought tolerance at the vegetative stage identified MDIL 2 and MDIL 11III as drought tolerant based on visual scoring. A regulated increase in stomatal conductance of MDIL 2 under drought stress indicates better water use efficiency that allowed it to survive for 7 days under 0% moisture level.

Integrated morpho-biochemical and transcriptome analyses reveal multidimensional response of upland cotton (Gossypium hirsutum L.) to low temperature stress during seedling establishment.

Cotton is a tropical/subtropical crop and is innately susceptible to cold. Using an approach that integrates morphological, biochemical, and transcriptome analyses, the study aimed to understand the molecular underpinnings of phenotypic adjustments in cotton seedlings under cold stress. Exposure of six cotton accessions to 15°C during the seedling stage significantly reduced chlorophyll content, stomatal conductance, plant height, and biomass, but increased malondialdehyde and proline production. Comparative transcriptome profiling of the cold-sensitive accession SA 3781 grown under low and normal temperatures showed the upregulation of genes related to the production of reactive oxygen species (ROS) under cold stress. Despite a similar upregulation of genes encoding metabolites that can scavenge ROS and provide osmoprotection for the cell, the stressed plants still exhibited oxidative stress in terms of lipid peroxidation. This may be due in part to the upregulation of abscisic acid synthesis genes and downregulation of chlorophyll synthesis genes effecting lower stomatal conductance and chlorophyll contents, respectively. Additionally, stomatal closure which is required to avoid the cooling effect and dehydration under cold conditions may have contributed in reducing the net photosynthetic rates in plants exposed to low temperature. These findings provide an insight into the expression of key genes regulating the phenotypic changes observed in cotton in response to cold stress.

Development of a PCR-based, genetic marker resource for the tomato-like nightshade relative, Solanum lycopersicoides using whole genome sequence analysis.

Solanum lycopersicoides is a wild nightshade relative of tomato with known resistance to a wide range of pests and pathogens, as well as tolerance to cold, drought and salt stress. To effectively utilize S. lycopersicoides as a genetic resource in breeding for tomato improvement, the underlying basis of observable traits in the species needs to be understood. Molecular markers are important tools that can unlock the genetic underpinnings of phenotypic variation in wild crop relatives. Unfortunately, DNA markers that are specific to S. lycopersicoides are limited in number, distribution and polymorphism rate. In this study, we developed a suite of S. lycopersicoides-specific SSR and indel markers by sequencing, building and analyzing a draft assembly of the wild nightshade genome. Mapping of a total of 1.45 Gb of S. lycopersicoides contigs against the tomato reference genome assembled a moderate number of contiguous reads into longer scaffolds. Interrogation of the obtained draft yielded SSR information for more than 55,000 loci in S. lycopersicoides for which more than 35,000 primers pairs were designed. Additionally, indel markers were developed based on sequence alignments between S. lycopersicoides and tomato. Synthesis and experimental validation of 345 primer sets resulted in the amplification of single and multilocus targets in S. lycopersicoides and polymorphic loci between S. lycopersicoides and tomato. Cross-species amplification of the 345 markers in tomato, eggplant, silverleaf nightshade and pepper resulted in varying degrees of transferability that ranged from 55 to 83%. The markers reported in this study significantly expands the genetic marker resource for S. lycopersicoides, as well as for related Solanum spp. for applications in genetics and breeding studies.

A novel locus from the wild allotetraploid rice species Oryza latifolia Desv. confers bacterial blight (Xanthomonas oryzae pv. oryzae) resistance in rice (O. sativa).

Bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo) is a major limiting factor to rice productivity worldwide. Genetic control through the identification of novel sources of bacterial blight resistance and their utilization in resistance breeding remains the most effective and economical strategy to manage the disease. Here we report the identification of a novel locus from the wild Oryza species, Oryza latifolia, conferring a race-specific resistance to Philippine Xoo race 9A (PXO339). The locus was identified from two introgression lines i.e. WH12-2252 and WH12-2256 that segregated from O. latifolia monosomic alien addition lines (MAALs). The discrete segregation ratio of susceptible and resistant phenotypes in the F2 (χ2[3:1] = 0.22 at p>0.05) and F3 (χ2[3:1] = 0.36 at p>0.05) populations indicates that PXO339 resistance in the MAAL-derived introgression lines (MDILs) is controlled by a single, recessive gene. Genotyping of a total of 216 F2, 1130 F3 and 288 F4 plants derived from crossing either of the MDILs with the recurrent parent used to generate the MAALs narrowed the candidate region to a 1,817 kb locus that extends from 10,425 to 12,266 kb in chromosome 12. Putative candidate genes that were identified by data mining and comparative sequence analysis can provide targets for further studies on mapping and cloning of the causal gene for PXO339 resistance in the MDILs. To our knowledge, this is the first report of a genetic locus from the allotetraploid wild rice, O. latifolia conferring race-specific resistance to bacterial blight.