Characterization of the ZCTs, a subgroup of Cys2-His2 zinc finger transcription factors regulating alkaloid biosynthesis in Catharanthus roseus.

KEY MESSAGE: The C. roseus ZCTs are jasmonate-responsive, can be induced by CrMYC2a, and can act as significant regulators of the terpenoid indole alkaloid pathway when highly expressed. Catharanthus roseus is the sole known producer of the anti-cancer terpenoid indole alkaloids (TIAs), vinblastine and vincristine. While the enzymatic steps of the pathway have been elucidated, an understanding of its regulation is still emerging. The present study characterizes an important subgroup of Cys2-His2 zinc finger transcription factors known as Zinc finger Catharanthus Transcription factors (ZCTs). We identified three new ZCT members (named ZCT4, ZCT5, and ZCT6) that clustered with the putative repressors of the TIA pathway, ZCT1, ZCT2, and ZCT3. We characterized the role of these six ZCTs as potential redundant regulators of the TIA pathway, and their tissue-specific and jasmonate-responsive expression. These ZCTs share high sequence conservation in their two Cys2-His2 zinc finger domains but differ in the spacer length and sequence between these zinc fingers. The transient overexpression of ZCTs in seedlings significantly repressed the promoters of the terpenoid (pLAMT) and condensation branch (pSTR1) of the TIA pathway, consistent with that previously reported for ZCT1, ZCT2, and ZCT3. In addition, ZCTs significantly repressed and indirectly activated several promoters of the vindoline pathway (not previously studied). The ZCTs differed in their tissue-specific expression but similarly increased with jasmonate in a dosage-dependent manner (except for ZCT5). We showed significant activation of the pZCT1 and pZCT3 promoters by the de-repressed CrMYC2a, suggesting that the jasmonate-responsive expression of the ZCTs can be mediated by CrMYC2a. In summary, the C. roseus ZCTs are jasmonate-responsive, can be induced by CrMYC2a, and can act as significant regulators of the TIA pathway when highly expressed.

Engineering the plant intracellular immune receptor Sr50 to restore recognition of the AvrSr50 escape mutant.

Sr50, an intracellular nucleotide-binding leucine-rich repeat receptor (NLR), confers resistance of wheat against stem rust caused by the fungal pathogen Puccinia graminis f. sp. tritici. The receptor recognizes the pathogen effector AvrSr50 through its C-terminal leucine-rich repeat domain, initiating a localized cell death immune response. However, this immunity is compromised by mutations in the effector, as in the escape mutant AvrSr50QCMJC, which evades Sr50 detection. In this study, we employed iterative computational structural analyses and site-directed mutagenesis for rational engineering of Sr50 to gain recognition of AvrSr50QCMJC. Following an initial structural hypothesis driven by molecular docking, we identified the Sr50K711D single mutant, which induces an intermediate immune response against AvrSr50QCMJC without losing recognition against AvrSr50. Increasing gene expression with a stronger promoter enabled the mutant to elicit a robust response, indicating weak effector recognition can be complemented by enhanced receptor expression. Further structural refinements led to the creation of five double mutants and two triple mutants with dual recognition of AvrSr50 and AvrSr50QCMJC with greater immune response intensities than Sr50K711D against the escape mutant. All effective mutations against AvrSr50QCMJC required the K711D substitution, indicating that multiple solutions exist for gain of recognition, but the path to reach these mutations may be confined. Furthermore, this single substitution alters the prediction of AlphaFold 2, allowing it to model the complex structure of Sr50K711D and AvrSr50 that match our final structural hypothesis. Collectively, our study outlines a framework for rational engineering of NLR systems to overcome pathogen escape mutations and provides datasets for future computational models for NLR resurrection.