Agroinfiltration for transient gene expression and characterisation of fungal pathogen effectors in cool-season grain legume hosts

Abstract

Key message Modified pEAQ-HT-DEST1 vectors were used for agroinfiltration in legumes. We demonstrate protein expression and export in pea, lentil, and faba bean; however, the method for chickpea was not successful. Abstract Agroinfiltration is a valuable research method for investigating virulence and avirulence effector proteins from pathogens and pests, where heterologous effector proteins are transiently expressed in plant leaves and hypersensitive necrosis responses and other effector functions can be assessed. Nicotiana benthamiana is widely used for agroinfiltration and the characterisation of broad-spectrum effectors. The method has also been used in other plant species including field pea, but not yet developed for chickpea, lentil, or faba bean. Here, we have modified the pEAQ-HT-DEST1 vector for expression of 6 × histidine-tagged green-fluorescent protein (GFP) and the known necrosis-inducing broad-spectrum effector necrosis and ethylene-inducing peptide (Nep1)-like protein (NLP). Modified pEAQ-based vectors were adapted to encode signal peptide sequences for apoplast targeting of expressed proteins. We used confocal microscopy to assess the level of GFP expression in agroinfiltrated leaves. While at 3 days after infiltration in N. benthamiana, GFP was expressed at a relatively high level, expression in field pea and faba bean at the same time point was relatively low. In lentil, an expression level of GFP similar to field pea and faba bean at 3 days was only observed after 5 days. Chickpea leaf cells were transformed at low frequency and agroinfiltration was concluded to not be successful for chickpea. We concluded that the pEAQ vector is suitable for testing host-specific effectors in field pea, lentil, and faba bean, but low transformation efficiency limits the utility of the method for chickpea.

A high-efficiency Agrobacterium-mediated transient expression system in the leaves of Artemisia annua L.

“…Besides, GUS activity in transgenic chrysanthemums using the CaMV35S promoter was low [ 55 ]. Similar results were also observed in chickpea [ 56 ]. Notably, along with the rapid development of DNA assembly technologies, multigene transformation which has the ability to introduce more complex and ambitious phenotypes into transgenic plants, is becoming more and more frequently-used in plant biotechnology [ 57 ].…”

Simple and efficient heterologous expression of necrosis‐inducing effectors using the model plant Nicotiana benthamiana

“…This method may also be an alternative for researchers who struggle with expressing SCR effectors using microbial expression systems. In line with this, a recent study showed that using agroinfiltration, the apoplast targeted NLP2 (necrosis and ethylene-inducing peptide-like protein) from Peyronellaea pinodes was successfully expressed and induced necrosis in legume plants including field pea, faba bean and lentil, but not in chickpea (Debler et al, 2021). This further corroborates that plants can serve as a heterologous expression system to produce biologically active fungal effectors in secreted form.…”

The novel avirulence effector AlAvr1 from Ascochyta lentis mediates host cultivar specificity of ascochyta blight in lentil

“…The host cultivar responses shown are representative of at least three replicates and additional images for other replicates are provided in Figure S5. Agroinfiltration with a negative control, green fluorescent protein (GFP), and a positive necrosis control, necrosis-inducing effector nep1-like protein (NLP) (Bailey, 1995;Debler et al, 2021), produced the expected responses in the lentil cultivars tested. (P94-24) and AlAvr1-2 (AlKewell).…”

“…To test the functional properties of the AlAvr1 effector biparental population trait-associated locus, we developed constructs based on the pEAQ-HT vector (Sainsbury & Lomonossoff, 2008;Sainsbury et al, 2009) for transient expression in lentil using agroinfiltration as described by Debler et al (2021). Vector diagrams are shown in Three-week-old lentil plants were infiltrated with Agrobacterium suspension at the adaxial leaf surface using a 1-ml plastic syringe.…”

“…A. lentis is from the family Didymellaceae, within the Pleosporales, and provides an additional plant disease model to research molecular mechanisms of pathogen-host interaction that contrasts with the pathogens from cereal and oilseed crops described above. A near-complete genome assembly for A. lentis has been recently published (Lee et al, 2021), and agroinfiltration, genetic transformation, and gene knockout protocols are available for research in the lentil-ascochyta pathosystem (Debler & Henares, 2020;Debler et al, 2021;Henares et al, 2019). However, so far very limited information is available about the pathogen-host interaction at the molecular level.…”