Functional Characterization of Monoterpenoid Indole Alkaloid (MIA) Biosynthetic Genes in Catharanthus roseus

The monoterpenoid indole alkaloids (MIAs) of Madagascar periwinkle (Catharanthus roseus) are known to be among the most important source of natural drugs used in various cancer chemotherapies. MIAs are derived by combining the iridoid secologanin with tryptamine to form the central precursor strictosidine that is then converted to most known MIAs, such as catharanthine and vindoline that dimerize to form anticancer vinblastine and vincristine. While their assembly is still poorly understood, the complex multistep pathways involved occur in several specialized cell types within leaves that are regulated by developmental and environmental cues. The organization of MIA pathways is also coupled to secretory mechanisms that allow the accumulation of catharanthine in the waxy leaf surface, separated from vindoline found within leaf cells. While the spatial separation of catharanthine and vindoline provides an explanation for the low levels of dimeric MIAs found in the plants, the secretion of catharanthine to the leaf surface is shown to be part of plant defense mechanisms against fungal infection and insect herbivores. The transcriptomic databases of Catharanthus roseus and various MIA producing plants are facilitating bioinformatic approaches to identify novel MIA biosynthetic genes. Virus-induced gene silencing (VIGS) is being used to screen these candidate genes for their involvement in iridoid biosynthesis pathway, especially in the identification of 7-deoxyloganic acid 7-hydroxylase (CrDL7H) shown by the accumulation of its substrate, 7-deoxyloganic acid and decreased level of secologanin along with catharanthine and vindoline. VIGS can also confirm the biochemical function of genes being identified, such as in the glucosylation of 7-deoxyloganetic acid by CrUGT8 shown by decreased level of secologanin and MIAs within silenced plants. Silencing of other iridoid biosynthetic genes, loganic acid O-methyltransferase (LAMT) and secologanin synthase (SLS) also confirm the metabolic route for iridoid biosynthesis in planta through 7-deoxyloganic acid, loganic acid, and loganin intermediates. This route is validated by high substrate specificity of CrUGT8 for 7-deoxyloganetic acid and CrDL7H for 7-deoxyloganic acid. Further localization studies of CrUGT8 and CrDL7H also show that these genes are preferentially expressed within Catharanthus leaves rather than in epidermal cells where the last two steps of secologanin biosynthesis occur.

The Molecular Consequences of CK2-mediated Phosphorylation of the TGA2 Transcription Factor within Systemic Acquired Resistance of Arabidopsis thaliana

During infection, the model plant Arabidopsis thaliana is capable of activating long lasting defence responses both in tissue directly affected by the pathogen and in more distal tissue. Systemic acquired resistance (SAR) is a type of systemic defence response deployed against biotrophic pathogens resulting in altered plant gene expression and production of antimicrobial compounds. One such gene involved in plant defence is called pathogenesis-related 1 (PR1) and is under the control of several protein regulators. TGA II-clade transcription factors (namely TGA2) repress PR1 activity prior to infection by forming large oligomeric complexes effectively blocking gene transcription. After pathogen detection, these complexes are dispersed by a mechanism unknown until now and free TGA molecules interact with the non-expressor of pathogenesis-related gene 1 (NPR1) protein forming an activating complex enabling PR1 transcription. This study elucidates the TGA2 dissociation mechanism by introducing protein kinase CK2 into this process. This enzyme efficiently phosphorylates TGA2 resulting in two crucial events. Firstly, the DNA-binding ability of this transcription factor is completely abolished explaining how the large TGA2 complexes are quickly evicted from the PR1 promoter. Secondly, a portion of TGA2 molecules dissociate from the complexes after phosphorylation which likely makes them available for the formation of the TGA2-NPR1 activating complex. We also show that phosphorylation of a multiserine motif found within TGA2’s N terminus is responsible for the change of affinity to DNA, while modification of a single threonine in the leucine zipper domain seems to be responsible for deoligomerization. Despite the substantial changes caused by phosphorylation, TGA2 is still capable of interacting with NPR1 and these proteins together form a complex on DNA promoting PR1 transcription. Therefore, we propose a change in the current model of how PR1 is regulated by adding CK2 which targets TGA2 displacing it’s complexes from the promoter and providing solitary TGA2 molecules for assembly of the activating complex. Amino acid sequences of regions targeted by CK2 in Arabidopsis TGA2 are similar to those found in TGA2 homologs in rice and tobacco. Therefore, the molecular mechanism that we have identified may be conserved among various plants, including important crop species, adding to the significance of our findings.

Unique and unifying themes in the mechanisms regulating the expression of the arabidopsis thaliana PR-1 and Solanum tuberosum PR-10a inducible defense genes

Arabidopsis is a model plant used to study disease resistance; Solanum tuberosum or potato is a crop species. Both plants possess inducible defense mechanisms that are deployed upon recognition of pathogen invasion. Transcriptional reprogramming is crucial to the activation of defense responses. The Pathogenesis-Related (PR) genes are activated in these defense programs. Expression of Arabidopsis PR-l and potato PR-10a serve as markers for the deployment of defense responses in these plants. PR-l expression indicates induction of systemic acquired resistance (SAR). Activation of SAR requires accumulation of salicylic acid (SA), in addition to the interaction of the non-expressor of pathogenesis-related genes I (NPRI), with the TGA transcription factors. The PR-10a is activated in response to pathogen invasion, wounding and elicitor treatment. PR-10a induction requires recruitment of the Whirly I (Whyl) activator to the promoter. This locus is also negatively regulated by the silencer element binding factor (SEBF). We established that both the PR-l and PR-10a are occupied by repressors under non-inducing conditions. TGA2 was found to be a constitutive resident and repressor of PR-l, which mediates repression by forming an oligomeric complex on the promoter. The DNA-binding activity of this oligomer required the TGA2 N-terminus (NT). Under resting conditions we determined that the PR-10a is bound by a repressosome containing SEBF and curiously the activator Pto interacting protein 4 (Pti4). In the context of this repressosome, SEBF is responsible for PR-10a binding, yet rWe also showed that PR-l and PR-10a are activated by different means. In PR-l activation the NPRI NT domain alleviates TGA2-mediated repression by interacting with the TGA2 NT. TGA2 remains at the PR-l but adopts a dimeric conformation and forms an enhanceosome with NPRl. In contrast, the PR-10a is activated by evicting the repressosome and recruiting Why! to the promoter. These results advance our understanding of the mechanisms regulating PR-l and PR-10a expression under resting and inducing conditions. This study also revealed that the means of regulation for related genes can differ greatly between model and crop s

The Arabidopsis NPR1 Protein Is a Receptor for the Plant Defense Hormone Salicylic Acid

Systemic Acquired Resistance (SAR) is a type of plant systemic resistance occurring against a broad spectrum of pathogens. It can be activated in response to pathogen infection in the model plant Arabidopsis thaliana and many agriculturally important crops. Upon SAR activation, the infected plant undergoes transcriptional reprogramming, marked by the induction of a battery of defense genes, including Pathogenesis-related (PR) genes. Activation of the PR-1 gene serves as a molecular marker for the deployment of SAR. The accumulation of a defense hormone, salicylic acid (SA) is crucial for the infected plant to mount SAR. Increased cellular levels of SA lead to the downstream activation of the PR-1 gene, triggered by the combined action of the Non-expressor of Pathogenesis-related Gene 1 (NPR1) protein and the TGA II-clade transcription factor (namely TGA2). Despite the importance of SA, its receptor has remained elusive for decades. In this study, we demonstrated that in Arabidopsis the NPR1 protein is a receptor for SA. SA physically binds to the C-terminal transactivation domain of NPR1. The two cysteines (Cys521 and Cys529), which are important for NPR1’s coactivator function, within this transactivation domain are critical for the binding of SA to NPR1. The interaction between SA and NPR1 requires a transition metal, copper, as a cofactor. Our results also suggested a conformational change in NPR1 upon SA binding, releasing the C-terminal transactivation domain from the N-terminal autoinhibitory BTB/POZ domain. These results advance our understanding of the plant immune function, specifically related to the molecular mechanisms underlying SAR. The discovery of NPR1 as a SA receptor enables future chemical screening for small molecules that activate plant immune responses through their interaction with NPR1 or NPR1-like proteins in commercially important plants. This will help in identifying the next generation of non-biocidal pesticides.