The rice SUB1A-1 gene, which encodes a group VII ethylene response factor (ERFVII), plays a pivotal role in rice survival under flooding stress, as well as other abiotic stresses. In Arabidopsis, five ERFVII factors play roles in regulating hypoxic responses. A characteristic feature of Arabidopsis ERFVIIs is a destabilizing N terminus, which functions as an N-degron that targets them for degradation via the oxygen-dependent N-end rule pathway of proteolysis, but permits their stabilization during hypoxia for hypoxia-responsive signaling. Despite having the canonical N-degron sequence, SUB1A-1 is not under N-end rule regulation, suggesting a distinct hypoxia signaling pathway in rice during submergence. Herein we show that two other rice ERFVIIs gene, ERF66 and ERF67, are directly transcriptionally up-regulated by SUB1A-1 under submergence. In contrast to SUB1A-1, ERF66 and ERF67 are substrates of the N-end rule pathway that are stabilized under hypoxia and may be responsible for triggering a stronger transcriptional response to promote submergence survival. In support of this, overexpression of ERF66 or ERF67 leads to activation of anaerobic survival genes and enhanced submergence tolerance. Furthermore, by using structural and protein-interaction analyses, we show that the C terminus of SUB1A-1 prevents its degradation via the N-end rule and directly interacts with the SUB1A-1 N terminus, which may explain the enhanced stability of SUB1A-1 despite bearing an N-degron sequence. In summary, our results suggest that SUB1A-1, ERF66, and ERF67 form a regulatory cascade involving transcriptional and N-end rule control, which allows rice to distinguish flooding from other SUB1A-1–regulated stresses.

Spermatozoa produced from spermatogonial stem cells (SSCs) are the vehicle by which genes of a male are passed to the next generation. A single SSC has the ability to self-renew and produce thousands of spermatozoa; therefore, it is an ideal target for genetic modification to efficiently generate transgenic animals in mammalian species. Rats are an important model organism for biological research; however, gene function studies have been difficult because of a limited ability to generate transgenic animals. Transgenic rat production through SSCs offers a means to overcome this obstacle. Because SSCs divide slowly both in vivo and in vitro, lentiviral vectors may be an ideal method for introducing stable genetic modification. Using a lentiviral vector, an enhanced green fluorescent protein (eGFP) transgene was introduced into the genome of cultured rat SSCs, which were microinjected into testes of immunodeficient mice to assess transduction efficiency. Approximately 40% of rat SSCs exposed to the lentiviral vector overnight carried the eGFP transgene and generated colonies of spermatogenesis. When transduced SSCs were transplanted into recipient rat testes, in which endogenous germ cells had been decreased but not eliminated by busulfan treatment, approximately 6% of offspring were transgenic. The transgene was stably integrated into the donor SSC genome and transmitted to and expressed by progeny in subsequent generations. Thus, lentiviral transduction of SSCs followed by transplantation is an effective means for generating transgenic rats through the male germline, and this approach may be applicable to other species in which existing methods are inadequate or not applicable.