Agroinfiltration and PVX Agroinfection in Potato and <em>Nicotiana benthamiana</em>

Abstract

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most commonly used agent for agroinfiltration is Agrobacterium tumefaciens, a pathogen of many dicot plant species. This implies that agroinfiltration can be applied to many plant species. Here, we present our protocols and expected results when applying these methods to the potato (Solanum tuberosum), its related wild tuber-bearing Solanum species (Solanum section Petota) and the model plant Nicotiana benthamiana. In addition to functional analysis of single genes, such as resistance (R) or avirulence (Avr) genes, the agroinfiltration assay is very suitable for recapitulating the R-AVR interactions associated with specific host pathogen interactions by simply delivering R and Avr transgenes into the same cell. However, some plant genotypes can raise nonspecific defense responses to Agrobacterium, as we observed for example for several potato genotypes. Compared to agroinfiltration, detection of AVR activity with PVX agroinfection is more sensitive, more high-throughput in functional screens and less sensitive to nonspecific defense responses to Agrobacterium. However, nonspecific defense to PVX can occur and there is a risk to miss responses due to virus-induced extreme resistance. Despite such limitations, in our experience, agroinfiltration and PVX agroinfection are both suitable and complementary assays that can be used simultaneously to confirm each other's results.

Tracking disease resistance deployment in potato breeding by enrichment sequencing

“…Similarly, effector recognition studies are dependent on the discovery of cognate avirulence genes which are not available for all NLRs, are limited by suitable expression systems that work in all host genotypes, and can suffer from a lack of specific plant responses (Vleeshouwers et al ., ). For example, the presence of Rx in some varieties including Cara and Picasso precludes the use of the often preferred PVX Agroinfection assay (Du et al ., ). Similarly, as shown for the cross between Alouette and Vitalia, some plants are rather recalcitrant to A. tumefaciens ‐based effector delivery (Table S3).…”

Genetic Engineering for Disease Resistance in Plants: Recent Progress and Future Perspectives

“…The hypersensitive response is often used as a marker to screen diverse plant germplasm for novel functional NLRs recognizing known effectors (Vleeshouwers and Oliver, 2014). During the screening, core virulence factors are delivered into plant leaves either as transiently expressed genes through Agro-infiltration (Du et al, 2014) or as genes expressed in laboratory bacterial strains with a functional secretion system (Zhang and Coaker, 2017).…”

Two distinct spontaneous mutations involved in white flower development in Lilium speciosum

“…An agroinfiltration assay was performed according to the method of Du et al (2014). Briefly, Agrobacterium strains harbouring different constructs were grown overnight in 10 mL of YEB medium (5 g/L sucrose, 5 g/L beef extract, 5 g/L peptone, 1 g/L yeast extract, and 2 mM MgSO 4 ) containing 50 lg/mL kanamycin, 20 lM acetosyringone, and 10 mM MES buffer (pH 5.6).…”