The Use of Agroinfiltration for Transient Expression of Plant Resistance and Fungal Effector Proteins in Nicotiana benthamiana Leaves

Ma, Lisong; Łukasik, Ewa; Gawehns, Fleur; Takken, Frank L. W.

doi:10.1007/978-1-61779-501-5_4

Cited by 134 publications

(128 citation statements)

References 34 publications

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“…Transient expression by agroinfiltration in Nicotiana benthamiana and N. tabacum was performed according to the protocols described by Ma et al (2012) and Dugdale et al (2014), respectively, with modifications. Briefly, we added a wash/recovery step where overnight Agrobacterium tumefaciens cultures in Luria broth medium containing appropriate antibiotics were harvested by centrifugation at 3000g for 5 min, followed by resuspension in fresh Luria broth medium without antibiotics and incubation for 30 min to 1 h at 28°C with 200 rpm shaking.…”

Section: Transient Protein Expression Assays In Nicotiana Benthamianamentioning

confidence: 99%

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Multiple Avirulence Loci and Allele-Specific Effector Recognition Control thePm3Race-Specific Resistance of Wheat to Powdery Mildew

et al. 2015

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In cereals, several mildew resistance genes occur as large allelic series; for example, in wheat (Triticum aestivum and Triticum turgidum), 17 functional Pm3 alleles confer agronomically important race-specific resistance to powdery mildew (Blumeria graminis). The molecular basis of race specificity has been characterized in wheat, but little is known about the corresponding avirulence genes in powdery mildew. Here, we dissected the genetics of avirulence for six Pm3 alleles and found that three major Avr loci affect avirulence, with a common locus_1 involved in all AvrPm3-Pm3 interactions. We cloned the effector gene AvrPm3 a2/f2 from locus_2, which is recognized by the Pm3a and Pm3f alleles. Induction of a Pm3 alleledependent hypersensitive response in transient assays in Nicotiana benthamiana and in wheat demonstrated specificity. Gene expression analysis of Bcg1 (encoded by locus_1) and AvrPm3 a2/f2 revealed significant differences between isolates, indicating that in addition to protein polymorphisms, expression levels play a role in avirulence. We propose a model for race specificity involving three components: an allele-specific avirulence effector, a resistance gene allele, and a pathogenencoded suppressor of avirulence. Thus, whereas a genetically simple allelic series controls specificity in the plant host, recognition on the pathogen side is more complex, allowing flexible evolutionary responses and adaptation to resistance genes.

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Section: Transient Protein Expression Assays In Nicotiana Benthamianamentioning

confidence: 99%

“…In N. tabacum, HR development was assessed 5 to 7 d after agroinfiltration. In N. benthamiana, HR development was revealed 5 d after infiltration by Trypan blue staining as described by Ma et al (2012).…”

Section: Transient Protein Expression Assays In Nicotiana Benthamianamentioning

confidence: 99%

Multiple Avirulence Loci and Allele-Specific Effector Recognition Control thePm3Race-Specific Resistance of Wheat to Powdery Mildew

et al. 2015

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“…To facilitate functional tests for an inserted gene, binary vectors that contain the PVX genome flanked by the Cauliflower mosaic virus 35S promoter and the nopaline synthase terminator, were cloned into the T-DNA of A. tumefaciens 20 3,4 . We also use Nicotiana benthamiana, which is renowned as a model plant in Solanaceous plants 14,21,22 . 1.…”

Section: Introductionmentioning

confidence: 99%

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

Rietman

Vleeshouwers

2014

JoVE

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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.

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“…However, Agrobacterium -mediated transient expression (Agroinfiltration) is an alternative and quick approach for validating a candidate R gene (Vaghchhipawala et al, 2011, Ma et al, 2012. It is a rapid and simple technique to study gene function and host-pathogen interaction and protein-protein interaction (Bendahmane et al, 2000, Wroblewski et al, 2005, Dodds et al, 2006 …”

Section: Molecular Interaction Between Brassica and L Maculansmentioning

confidence: 99%

Genome wide identification of NBS-LRR genes in Brassica and their association with disease resistance in Brassica napus

Alamery¹

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Brassica napus (canola/rapeseed/oilseed rape) is an important commercial oilseed crop in Australia with an annual production of approximately 1.6 million tons of oil. Canola is an important source of edible vegetable oil and has a broad range of industrial purposes. Blackleg disease (stem canker) caused by the fungal pathogen Leptosphaeria maculans, is one of the most devastating diseases in B. napus. This disease causes significant yield losses with an annual average loss of 15 to 48 %, although losses can reach up to 80% worldwide, mainly in Europe, Australia and Canada.In order to develop an effective strategy to control the blackleg disease, there is a need to identify blackleg resistance genes in Brassica species and understand the genetic interaction between plant resistance genes and the pathogen avirulence genes. Thus, identification of Nucleotide Binding SiteLeucine Rich Repeat (NBS-LRR) resistance genes is one of the most important objectives of understanding resistance. NBS-LRR resistance genes have been extensively studied because they represent the largest class of disease resistance genes and play a critical role in defending plants from pathogens.The objective of this study was to perform comprehensive analysis on identification and characterization of NBS-LRR genes in the B. napus genome and to study the synteny and conservation of NBS-LRR genes between Brassica species. In this study, a total of 641, 249 and 443 NBS-LRR encoding genes in B. napus, B. rapa and B. oleracea, respectively, were identified.The comparative analysis between B. napus and its progenitor species indicated that NBS-LRR genes exhibited similar gene structure, genomic location, arrangement in clusters and syntenic relationships. The results provide evidence that there was a selective advantage to maintaining similar features of NBS-LRR genes in B. napus to both B. rapa and B. oleracea following polyploidization. More than 60% of NBS LRR genes from the progenitor species were conserved.The differences in NBS-LRR gene conservation could be attributed to gene losses or selection pressure to offer species-specific or cultivars-specific resistance.This study found that the NBS-LRR resistance genes are physically clustered and individual genes involved in clusters were more polymorphic and subject to evolutionary process than singleton genes. These clusters, which have been described in many other species, provide a reservoir of genetic variation influenced by tandem duplication and selection pressure. In addition, there was a significant correlation and co-localization between the number of NBS-LRR genes within the disease QTL intervals and the number of genes involved in gene clusters or duplication. This ii correlation provides evidence that NBS-LRR are distributed and clustered throughout the genome and tends to be linked and associated with disease QTL intervals. Chalhoub, B., Denoeud, F., Liu, S., Parkin, I. A. P., Tang, H., Wang, X., Chiquet, J., Belcram, H., Tong, C., Samans, B., Corréa, M., Da Silva, C., Just, ...

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The Use of Agroinfiltration for Transient Expression of Plant Resistance and Fungal Effector Proteins in Nicotiana benthamiana Leaves

Cited by 134 publications

References 34 publications

Multiple Avirulence Loci and Allele-Specific Effector Recognition Control thePm3Race-Specific Resistance of Wheat to Powdery Mildew

Multiple Avirulence Loci and Allele-Specific Effector Recognition Control thePm3Race-Specific Resistance of Wheat to Powdery Mildew

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

Genome wide identification of NBS-LRR genes in Brassica and their association with disease resistance in Brassica napus

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