Summary: S‐RNase participates in at least three mechanisms of pollen rejection. It functions in S‐specific pollen rejection (self‐incompatibility) and in at least two distinct interspecific mechanisms of pollen rejection in Nicotiana. S‐specific pollen rejection and rejection of pollen from Nicotiana plumbaginifolia also require additional stylar proteins. Transmitting‐tract‐specific (TTS) protein, 120 kDa glycoprotein (120K) and pistil extensin‐like protein III (PELP III) are stylar glycoproteins that bind S‐RNase in vitro and are also known to interact with pollen. Here we tested whether these glycoproteins have a direct role in pollen rejection. 120K shows the most polymorphism in size between Nicotiana species. Larger 120K‐like proteins are often correlated with S‐specific pollen rejection. Sequencing results suggest that the polymorphism primarily reflects differences in glycosylation, although indels also occur in the predicted polypeptides. Using RNA interference (RNAi), we suppressed expression of 120K to determine if it is required for S‐specific pollen rejection. Transgenic SC N. plumbaginifolia × SI Nicotiana alata (S₁₀₅S₁₀₅ or S_C10S_C10) hybrids with no detectable 120K were unable to perform S‐specific pollen rejection. Thus, 120K has a direct role in S‐specific pollen rejection. However, suppression of 120K had no effect on rejection of N. plumbaginifolia pollen. In contrast, suppression of HT‐B, a factor previously implicated in S‐specific pollen rejection, disrupts rejection of N. plumbaginifolia pollen. Thus, S‐specific pollen rejection and rejection of N. plumbaginifolia pollen are mechanistically distinct, because they require different non‐S‐RNase factors.

The current technologies to place new DNA into specific locations in plant genomes are low frequency and error-prone, and this inefficiency hampers genome-editing approaches to develop improved crops^1,2. Often considered to be genome ‘parasites’, transposable elements (TEs) evolved to insert their DNA seamlessly into genomes^3–5. Eukaryotic TEs select their site of insertion based on preferences for chromatin contexts, which differ for each TE type^6–9. Here we developed a genome engineering tool that controls the TE insertion site and cargo delivered, taking advantage of the natural ability of the TE to precisely excise and insert into the genome. Inspired by CRISPR-associated transposases that target transposition in a programmable manner in bacteria^10–12, we fused the rice Pong transposase protein to the Cas9 or Cas12a programmable nucleases. We demonstrated sequence-specific targeted insertion (guided by the CRISPR gRNA) of enhancer elements, an open reading frame and a gene expression cassette into the genome of the model plant Arabidopsis. We then translated this system into soybean—a major global crop in need of targeted insertion technology. We have engineered a TE ‘parasite’ into a usable and accessible toolkit that enables the sequence-specific targeting of custom DNA into plant genomes.

An active miniature inverted repeat transposable element (MITE), mPing , was discovered by computer-assisted analysis of rice genome sequence. The mPing element is mobile in rice cell culture and in a few rice strains where it has been amplified to >1,000 copies during recent domestication. However, determination of the transposase source and characterization of the mechanism of transposition have been hampered by the high copy number of mPing and the presence of several candidate autonomous elements in the rice genome. Here, we report that mPing is active in Arabidopsis thaliana , where its transposition is catalyzed by three sources of transposase from rice: the autonomous Ping and Pong elements and by a cDNA derived from a Ping transcript. In addition to transposase, the product of a second element-encoded ORF of unknown function is also required for mPing transposition. Excision of mPing in A. thaliana is usually precise, and transposed copies usually insert into unlinked sites in the genome that are preferentially in or near genes. As such, this will be a valuable assay system for the dissection of MITE transposition and a potentially powerful tagging system for gene discovery in eukaryotes.

Insertional mutagenesis of legume genomes such as soybean (Glycine max) should aid in identifying genes responsible for key traits such as nitrogen fixation and seed quality. The relatively low throughput of soybean transformation necessitates the use of a transposon-tagging strategy where a single transformation event will produce many mutations over a number of generations. However, existing transposon-tagging tools being used in legumes are of limited utility because of restricted transposition (Ac/Ds: soybean) or the requirement for tissue culture activation (Tnt1: Medicago truncatula). A recently discovered transposable element from rice (Oryza sativa), mPing, and the genes required for its mobilization, were transferred to soybean to determine if it will be an improvement over the other available transposon-tagging tools. Stable transformation events in soybean were tested for mPing transposition. Analysis of mPing excision at early and late embryo developmental stages revealed increased excision during late development in most transgenic lines, suggesting that transposition is developmentally regulated. Transgenic lines that produced heritable mPing insertions were identified, with the plants from the highest activity line producing at least one new insertion per generation. Analysis of the mPing insertion sites in the soybean genome revealed that features displayed in rice were retained including transposition to unlinked sites and a preference for insertion within 2.5 kb of a gene. Taken together these findings indicate that mPing has the characteristics necessary for an effective transposon-tagging resource.