Inhibition of in vitro pollen tube growth in ‘Hyuganatsu’ (Citrus tamurana hort. ex Tanaka) by recombinant S₁₅-RNase protein

The genus Citrus includes plants that display gametophytic self-incompatibility (GSI), with recent studies identifying S-RNase as the female factor controlling this GSI in citrus species. Citrus tamurana ‘Hyuganatsu’ is a local Japanese citrus that also shows GSI, with S₁₅-RNase believed to be located on one of the S alleles acting as the female S factor.
However, no direct evidence was presented whether this S₁₅-RNase has a pollen tube elongation inhibitory action.
To address this, we generated a recombinant protein of S₁₅-RNase and treated germinating pollen of ‘Hyuganatsu’ in vitro.
We isolated S₁₅-RNase cDNA from the style of ‘Hyuganatsu’ flower, and transformed it with maltose-binding protein (MBP) and histidine (His) tags into Escherichia coli. The expressed recombinant protein (MBP-S₁₅-RNase-His) was recovered from E. coli. A recombinant protein with only MBP and His tags without S₁₅-RNase (MBP-His) was also prepared.
The MBP-S₁₅-RNase-His recombinant protein degraded RNA extracted from ‘Hyuganatsu’ styles, indicating its RNA-degrading activity.
The MBP-S₁₅-RNase-His and MBP-His protein treatments both inhibited pollen tube elongation.
The average pollen tube lengths of MBP-S₁₅-RNase-His and MBP-His treatments were 67.43 and 83.65% compared to the control, respectively, significantly lower than those of the control.
These results suggest that S₁₅-RNase protein has an inhibitory effect on pollen tube elongation, confirming its role as the female factor controlling GSI in ‘Hyuganatsu’.

IV Asian Horticultural Congress – AHC2023

C. Honsho, T. Inaba, Y. Sugimoto, S. Ishimura, T. Tetsumura

self-incompatibility, recombinant protein, S-RNase, citrus

https://doi.org/10.17660/ActaHortic.2024.1404.84