Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana
Du, J. ; Rietman, H. ; Vleeshouwers, V.G.A.A. - \ 2014
Journal of Visualized Experiments (2014)83. - ISSN 1940-087X - 7 p.
late blight resistance - mediated plant transformation - phytophthora-infestans - pathogen phytophthora - disease resistance - effector proteins - gene-expression - binary vector - agrobacterium - recognition
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.
Tsw gene-based resistance is triggered by a functional RNA silencing suppressor protein of the Tomato spotted wilt virus
Ronde, D. de; Butterbach, P.B.E. ; Lohuis, H. ; Hedil, M. ; Lent, J.W.M. van; Kormelink, R.J.M. - \ 2013
Molecular Plant Pathology 14 (2013)4. - ISSN 1464-6722 - p. 405 - 415.
mediated plant transformation - capsicum-chinense - cell-death - disease-resistance - lycopersicon-esculentum - viral suppressors - sw-5 gene - potato - tospovirus - agrobacterium
As a result of contradictory reports, the avirulence (Avr) determinant that triggers Tsw gene-based resistance in Capsicum annuum against the Tomato spotted wilt virus (TSWV) is still unresolved. Here, the N and NSs genes of resistance-inducing (RI) and resistance-breaking (RB) isolates were cloned and transiently expressed in resistant Capsicum plants to determine the identity of the Avr protein. It was shown that the NSsRI protein triggered a hypersensitive response (HR) in Tsw-containing Capsicum plants, but not in susceptible Capsicum, whereas no HR was discerned after expression of the NRI/RB protein, or when NSsRB was expressed. Although NSsRI was able to suppress the silencing of a functional green fluorescence protein (GFP) construct during Agrobacterium tumefaciens transient assays on Nicotiana benthamiana, NSsRB had lost this capacity. The observation that RB isolates suppressed local GFP silencing during an infection indicated a recovery of RNA silencing suppressor activity for the NSs protein or the presence of another RNA interference (RNAi) suppressor. The role of NSs as RNA silencing suppressor and Avr determinant is discussed in the light of a putative interplay between RNAi and the natural Tsw resistance gene
Optimized agroinfiltration and virus-induced gene silencing to study Ve1-mediated Verticillium resistance in tobacco
Zhang, Z. ; Fradin, E. ; Jonge, R. de; Esse, P. van; Smit, P. ; Liu, C.M. ; Thomma, B.P.H.J. - \ 2013
Molecular Plant-Microbe Interactions 26 (2013)2. - ISSN 0894-0282 - p. 182 - 190.
receptor-like proteins - transient expression system - mediated plant transformation - functional-analysis - disease resistance - albo-atrum - hypersensitive response - nicotiana-benthamiana - arabidopsis-thaliana - binary vectors
Recognition of pathogen effectors by plant immune receptors often leads to the activation of a hypersensitive response (HR), which is a rapid and localized cell death of plant tissue surrounding the site at which recognition occurs. Due to its particular amenability to transient assays for functional genetics, tobacco is a model for immune signaling in the Solanaceae plant family. Here, we show that coexpression of the tomato (Solanum lycopersicum) immune receptor Ve1 and the corresponding Verticillium effector protein Ave1 leads to HR only in particular tobacco species. Whereas HR is obtained in Nicotiana tabacum, no such response is obtained in N. benthamiana. Furthermore, our analysis revealed an endogenous Ve1 ortholog in Nicotiana glutinosa, as expression of Ave1 in absence of Ve1 induced a HR, and N. glutinosa was found to be resistant against race 1 Verticillium dahliae. We furthermore report the establishment of virus-induced gene silencing in N. tabacum for functional analysis of Ve1 signaling. Collectively, our data show that N. tabacum can be used as a model plant to study Ve1-mediated immune signaling.
Phytophthora infestans isolates lacking class I ipiO variants are virulent on Rpi-blb1 potato
Champouret, N. ; Bouwmeester, K. ; Rietman, H. ; Lee, T. van der; Maliepaard, C.A. ; Heupink, A. ; Vondervoort, P.J.I. van de; Jacobsen, E. ; Visser, R.G.F. ; Vossen, E.A.G. van der; Govers, F. ; Vleeshouwers, V.G.A.A. - \ 2009
Molecular Plant-Microbe Interactions 22 (2009)12. - ISSN 0894-0282 - p. 1535 - 1545.
late blight resistance - mediated plant transformation - broad-spectrum resistance - nevado-de-toluca - solanum-bulbocastanum - central mexico - downy mildew - cell-death - population-structure - cultivated potato
A strategy to control the devastating late blight disease is providing potato cultivars with genes that are effective in resistance to a broad spectrum of Phytophthora infestans isolates. Thus far, most late blight resistance (R) genes that were introgressed in potato were quickly defeated. In contrast, the Rpi-blb1 gene originating from Solanum bulbocastanum has performed as an exclusive broad-spectrum R gene for many years. Recently, the RXLR effector family ipiO was identified to contain Avr-blb1. Monitoring the genetic diversity of the ipiO family in a large set of isolates of P. infestans and related species resulted in 16 ipiO variants in three distinct classes. Class I and class II but not class III ipiO variants induce cell death when coinfiltrated with Rpi-blb1 in Nicotiana benthamiana. Class I is highly diverse and is represented in all analyzed P. infestans isolates except two Mexican P. infestans isolates, and these were found virulent on Rpi-blb1 plants. In its C-terminal domain, IPI-O contains a W motif that is essential for triggering Rpi-blb1-mediated cell death and is under positive selection. This study shows that profiling the variation of Avr-blb1 within a P. infestans population is instrumental for predicting the effectiveness of Rpi-blb1-mediated resistance in potato.