Determining the GmRIN4 Requirements of the Soybean Disease Resistance Proteins Rpg1b and Rpg1r Using a <i>Nicotiana glutinosa</i>-Based Agroinfiltration System

<div><p><i>Rpg1b</i> and <i>Rpg1r</i> are soybean disease resistance (<i>R</i>) genes responsible for conferring resistance to <i>Pseudomonas syringae</i> strains expressing the effectors AvrB and AvrRpm1, respectively. The study of these cloned genes would be greatly facilitated by the availability of a suitable transient expression system. The commonly used <i>Niciotiana benthamiana</i>-based system is not suitable for studying <i>Rpg1b</i> and <i>Rpg1r</i> function, however, because expression of AvrB or AvrRpm1 alone induces a hypersensitive response (HR), indicating that <i>N. benthamiana</i> contains endogenous <i>R</i> genes that recognize these effectors. To identify a suitable alternative host for transient expression assays, we screened 13 species of <i>Nicotiana</i> along with 11 accessions of <i>N. tabacum</i> for lack of response to transient expression of <i>AvrB</i> and <i>AvrRpm1</i>. We found that <i>N. glutinosa</i> did not respond to either effector and was readily transformable as determined by transient expression of β-glucuronidase. Using this system, we determined that <i>Rpg1b</i>-mediated HR in <i>N. glutinosa</i> required co-expression of <i>avrB</i> and a soybean ortholog of the Arabidopsis <i>RIN4</i> gene. All four soybean <i>RIN4</i> orthologs tested worked in the assay. In contrast, Rpg1r did not require co-expression of a soybean <i>RIN4</i> ortholog to recognize AvrRpm1, but recognition was suppressed by co-expression with AvrRpt2. These observations suggest that an endogenous <i>RIN4</i> gene in <i>N. glutinosa</i> can substitute for the soybean <i>RIN4</i> ortholog in the recognition of AvrRpm1 by Rpg1r.</p>