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Madagascar periwinkle (Catharanthus roseus, family Apocynaceae) is a reservoir of more than 130 monoterpene indole alkaloids (MIAs) including the famous anti-neoplastic dimeric MIAs vinblastine and vincristine, and anti-hypertensive monomeric MIAs ajmalicine and serpentine. Understanding the biosynthetic steps and regulatory factors leading to the formation of MIAs is crucial for rational engineering to achieve targeted enhancement of different MIAs. Due to its highly recalcitrant nature, C. roseus is considered genetically non-tractable for transformation at the whole-plant level. Though few reports have demonstrated tissue culture-mediated regeneration and transformation of C. roseus at whole-plant level recently, the efficiency and reproducibility of these protocols have been a major challenge. To overcome this, we have developed a tissue-culture-independent Agrobacterium-mediated in planta transformation method in C. roseus. Using this method, we were able to efficiently generate stable transgenic plants without relying on the cumbersome methods of tissue-culture regeneration and transformation. Moreover, the transformed plants obtained through this in planta method exhibited stability in subsequent generations. Our method is useful not only for the elucidation of biosynthetic and regulatory steps involved in MIA formation through transgenic plant approach but also for metabolic engineering at the whole-plant level in C. roseus., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Virus-induced gene silencing (VIGS) is a functional genomics tool to transiently downregulate the expression of target gene(s) by exploiting the plant's innate defense mechanism against invading RNA viruses. VIGS is a rapid and efficient approach to analyze the gene function, particularly, in the plants that are not amenable to stable genetic transformation. This strategy has been successfully used to decipher the function of several genes and transcription factors involved in the biosynthesis of specialized metabolites and regulation of specialized metabolism, respectively, in different medicinal and aromatic plants. Here, we describe a detailed Tobacco rattle virus (TRV)-mediated VIGS protocol for silencing of the gene encoding Phytoene desaturase (PDS) in important medicinal plants Catharanthus roseus, Calotropis gigantean, Rauwolfia serpentina, and Ocimum basilicum. Our methods allow the study of gene function within 3-4 weeks after agro-inoculation, and can be an easy and efficient approach for future studies on understanding of the biosynthesis of specialized metabolites in these important medicinal plants., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Deglycosylation is a key step in the activation of specialized metabolites involved in plant defense mechanisms. This reaction is notably catalyzed by β-glucosidases of the glycosyl hydrolase 1 (GH1) family such as strictosidine β-d-glucosidase (SGD) from Catharanthus roseus. SGD catalyzes the deglycosylation of strictosidine, forming a highly reactive aglycone involved in the synthesis of cytotoxic monoterpene indole alkaloids (MIAs) and in the crosslinking of aggressor proteins. By exploring C. roseus transcriptomic resources, we identified an alternative splicing event of the SGD gene leading to the formation of a shorter isoform of this enzyme (shSGD) that lacks the last 71-residues and whose transcript ratio with SGD ranges from 1.7% up to 42.8%, depending on organs and conditions. Whereas it completely lacks β-glucosidase activity, shSGD interacts with SGD and causes the disruption of SGD multimers. Such disorganization drastically inhibits SGD activity and impacts downstream MIA synthesis. In addition, shSGD disrupts the metabolic channeling of downstream biosynthetic steps by hampering the recruitment of tetrahydroalstonine synthase in cell nuclei. shSGD thus corresponds to a pseudo-enzyme acting as a regulator of MIA biosynthesis. These data shed light on a peculiar control mechanism of β-glucosidase multimerization, an organization common to many defensive GH1 members., (© American Society of Plant Biologists 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Virus-induced gene silencing (VIGS) has emerged as a fast and efficient reverse and forward genetics tool to study gene function in model plants as well as in agriculturally important plants. In addition, VIGS approach has been successfully used to provide insights into the role of several genes and regulators involved in plant secondary metabolism. Ashwagandha (Withania somnifera) is an important Indian medicinal plant that accumulates pharmacologically important triterpenoid steroidal lactones, which are collectively termed as withanolides. W. somnifera being a highly recalcitrant plant for genetic transformation, Tobacco rattle virus (TRV)-mediated VIGS was established by our group to facilitate understanding of withanolides' pathway. Here, we describe a detailed procedure to carry out VIGS for gene function studies in W. somnifera.