Rhomboid-mediated cleavage of the immune receptor XA21 protects grain set and male fertility in rice

Satyam Vergish, Xiaoen Huang, Guiyun Zhang, Beatriz de Toledo Franceschi, Jian-Liang Li, Xiao-Xia Wu, Joana Nuraj, Jose C. Huguet-Tapia, Apekshya Parajuli, Xiuhua Chen, Ritu Shekhar, Dali Liu, Wu-Ming Xiao, Shijuan Dou, Guo-zhen Liu, Erica M. Goss, Liya Pi, Sixue Chen, Karen E. Koch, and Wen-Yuan Song

PNAS; May 30, 2025; 122 (22) e2502025122; https://doi.org/10.1073/pnas.2502025122

Significance

The protection of key developmental processes from the immune system, while allowing the immune system to defend against pathogens, is central for the survival and reproduction of nearly all multicellular organisms. The rice immune receptor XA21 specifies resistance against the bacterial pathogen Xanthomonas oryzae pv. oryzae. Here, we demonstrate that the rice rhomboid-like protease OsRBL3b controls the steady-state level of XA21 in spikelets and prevents XA21-induced male sterility and yield penalty. In leaves, the OsRBL3b gene is expressed at low levels and has little effect on XA21-mediated immunity. Therefore, these results reveal a regulatory mechanism by which rhomboid-mediated proteolysis of a transmembrane domain-containing receptor prevents detrimental impacts of an activated immune system on reproduction in a major food crop.

Abstract

To maintain growth and to successfully reproduce, organisms must protect key functions in specific tissues, particularly when countering pathogen invasion using internal defensive proteins that may disrupt their own developmental processes. The rice immune receptor XA21 confers race-specific resistance against Xanthomonas oryzae pv. oryzae, which causes the deadly disease bacterial leaf blight. Here, we demonstrate that XA21 is cleaved by the rhomboid-like protease OsRBL3b, likely within its transmembrane domain. OsRBL3b mRNA transcripts are preferentially expressed in rice spikelets. Rice plants expressing Xa21 but lacking a functional OsRBL3b displayed impaired anther dehiscence and pollen viability, resulting in male sterility and yield reduction with high levels of XA21 protein present in spikelets during anthesis. In leaves, osrbl3b mutants expressing XA21 had normal levels of this resistance protein and disease immunity. This balance between reproduction and disease resistance through the specific expression of a rhomboid protease may be key to limiting the detrimental effects of an active immune response and may be useful in future for genetic improvement of crops.

See https://www.pnas.org/doi/10.1073/pnas.2502025122

Figure 1

Expression of OsRBL3b and accumulation of XA21 in rice spikelets at anthesis. (A) OsRBL3b is preferentially expressed in spikelets. Levels of mRNA were quantified by RT-qPCR using the Myc-Xa21^L line. Results were normalized to the expression of the housekeeping genes UBIQUITIN5 and ACTIN. Expression levels in the indicated tissues were expressed relative to those of 2-mo-old leaves, which were arbitrarily set to 1 unit. Values are means ± SD of three biological replicates, each with three technical replicates. DAF, days after flowering. BA, before anthesis. (B) Accumulation of Myc-XA21 protein in spikelets. Myc-XA21 protein levels were determined in the indicated lines by immunoblot using an anti-Myc antibody, with ATPase levels as loading controls. WT^sib lacking XA21 is a negative control. Spikelets from more than three plants per line were used for protein extraction. (C) Quantification of Myc-XA21 from (B). This immunoblotting was repeated three times with similar results (all replicates in this study are shown in SI Appendix). In each experiment, the Myc-XA21 protein level was normalized against the ATPase level. The Myc-XA21 protein level in Myc-Xa21^L was set to 1.0 as references for calculating the relative level of Myc-XA21 in Myc-Xa21^H. Statistical analyses were performed using Student’s t-test. Asterisks denote statistically significant differences (*P < 0.05, **P < 0.01).