Milnes, L. | Search bioRxiv

bioRxiv2024

Interfamily co-transfer of sensor and helper NLRs extends immune receptor functionality between angiosperms

Plant nucleotide-binding, leucine-rich repeat (NLR) immune receptors recognize pathogen effectors and activate defense. NLR genes can be non-functional in distantly related plants (restricted taxonomic functionality, RTF). Here, we enable Solanaceae NLR gene function in rice, soybean and Arabidopsis by co-delivering sensor NLR genes with their cognate NRC-type helper NLRs. In soybean protoplasts and in Arabidopsis plants, Solanum americanum Rpi-amr1, Rpi-amr3 and pepper Bs2 sensor NLRs confer cognate effector responsiveness if co-expressed with NRC helper NLRs. Rice carrying pepper Bs2 and NRCs recognizes the conserved effector, AvrBs2, and resists an important pathogen, Xanthomonas oryzae pv. oryzicola for which no resistance gene is available in rice. Rice lines carrying sensor and helper NLR genes otherwise resemble wild-type, with unaltered basal resistance or field fitness. Thus, interfamily co-transfer of sensor and helper NLRs can broaden utility of sensor NLRs, extending the tools available to control diseases of rice, soybean, Brassicas and other crops.

plant biology↗

bioRxiv2023

pOPARA: Vectors for Golden Gate assembly of expression constructs containing the araBAD promoter

Heterologous protein production is often required to investigate the structural, biochemical, and biophysical properties of a protein of interest. Frequently, optimisation of expression conditions is required to obtain soluble protein and maximise yield. Trialling a variety of solubility and purification tags, as well as constructs containing different regulatory elements, is desirable. Golden Gate cloning allows modular assembly of different constructs using Type IIS restriction enzymes. The pOPIN vector suite, which utilises the T7 expression system, has been adapted to be compatible with Golden Gate assembly. Here, we present the pOPARA vectors. Expression from pOPARA vectors is driven by the araBAD promoter (pBAD) and is induced by addition of arabinose to the culture medium. pOPARA allows modular assembly of expression constructs using Golden Gate cloning with the CDS of interest and an optional C-terminal tag. pOPARA1 contains a carbenicillin resistance cassette flanked by restriction sites to allow exchange of the selectable markers. In pOPARA2, the carbenicillin resistance cassette has been exchanged for a spectinomycin resistance cassette. We demonstrate that both vectors can be used to express and produce a control protein.

molecular biology↗