Nicotiana benthamiana XYLEM CYSTEINE PROTEASE genes facilitate tracheary element formation in interfamily grafting

Abstract

Grafting is a plant propagation technique widely used in agriculture. A recent discovery of the capability of interfamily grafting in Nicotiana has expanded the potential combinations of grafting. In this study, we showed that xylem connection is essential for the achievement of interfamily grafting and investigated the molecular basis of xylem formation at the graft junction. Transcriptome and gene network analyses revealed gene modules for tracheary element (TE) formation during grafting that include genes associated with xylem cell differentiation and immune response. The reliability of the drawn network was validated by examining the role of the Nicotiana benthamiana XYLEM CYSTEINE PROTEASE (NbXCP) genes in TE formation during interfamily grafting. Promoter activities of NbXCP1 and NbXCP2 genes were found in differentiating TE cells in the stem and callus tissues at the graft junction. Analysis of a Nbxcp1;Nbxcp2 loss-of-function mutant indicated that NbXCPs control the timing of de novo TE formation at the graft junction. Moreover, grafts of the NbXCP1 overexpressor increased the scion growth rate as well as the fruit size. Thus, we identified gene modules for TE formation at the graft boundary and demonstrated potential ways to enhance Nicotiana interfamily grafting.

Acknowledgements

We thank M. Hattori, M. Matsumoto and F. Tobe for their technical assistance. We are grateful to C. Masuta (Hokkaido University, Japan) for providing CMV vectors. This work was supported by grants from the Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research (20H03273, 21H00368 and 21H05657 to MN and 22 K06181 to KK), Japan Science and Technology Agency (JPMJTR194G to MN), and China Scholarship Council (CSC; No. 201908050204 to CH).

Author contributions

CH, KK, and MN conceived the study and designed the experiments. CH performed the grafting experiments and microscopy analysis. CH and RT performed the VIGS experiments. CH and KK analyzed the transcriptome data. KK conducted the network analyses. NM conducted the yeast one-hybrid experiments. RS and KT performed radioisotope experiments. CH, KK, and MN wrote the manuscript.

Data availability

All relevant data and figures in this study can be found within the article and its supporting materials.

Conflict of interests

None declared.

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