Summary The intracellular nucleotide‐binding domain leucine‐rich repeat (NLR) class of immune receptors plays an important role in plant viral defence. Plant NLRs recognize viruses through direct or indirect association of viral proteins, triggering a downstream defence response to prevent viral proliferation and movement within the plant. This review focuses on current knowledge of intracellular perception of viral pathogens, activation of NLRs and the downstream signalling components involved in plant viral defence.

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Parasites target the plant immune system for successful colonization. A new study in PLOS Biology reveals that unrelated parasites have evolved effectors that specifically suppress the function of helper nucleotide-binding leucine-rich repeats (NLRs), explaining the complex plant-parasite coevolutionary dynamics.Plants have evolved a robust innate immune system to defend against a large number of parasites present in their ecological niche [1]. The plant's first layer of defense involves detection of conserved pathogen/microbe-associated molecular patterns (PAMPs/MAMPs) leading to pattern-triggered immunity (PTI). Parasites have evolved to sense and suppress PTI by secreting virulence factors called effectors into plant cells. Plants have evolved to recognize parasiteencoded effectors via the nucleotide-binding leucine-rich repeat (NLR) class of immune receptors and activate more robust effector-triggered immunity (ETI), which ultimately leads to cell death at the site of infection called the hypersensitive response (HR) [1] (Fig 1). This serves to starve out parasites and prevent further colonization. The plant genome encodes a large number of NLRs [2,3]. The NLRs that are involved in the recognition of parasite effectors either directly or indirectly are classified as sensor NLRs [2,4,5] (Fig 1). Accumulating evidence indicates that some sensor NLRs require the function of other NLRs, referred to as helper NLRs, to activate downstream immune signaling and cell death induction [4,5] (Fig 1). To date, there are 3 described helper NLR families: the Activated Disease Resistance 1 (ADR1) family, the N Requirement Gene 1 (NRG1) family, and NB-LRR protein required for HR-associated cell death (NRC) family [6].The Solanaceae-specific NRC family is required for the function of a large number of CCtype sensor NLRs that confer resistance to a diverse array of parasites [4,5]. An emerging model shows that NRCs and their sensor NLR partners form a complex genetic network, in which NRCs form redundant central nodes and also exhibit some specificity toward their sensor NLR partners. For example, the sensor NLR Prf requires NRC2 and NRC3 to confer resistance to Pseudomonas syringae bacteria, AU : Anabbreviationlisthasbeencompiledforthoseusedthrougho but NLR Rpi-blb2 only requires NRC4 to confer resistance to potato blight caused by the oomycete pathogen Phytophthora infestans [4,5]. Furthermore, Rx requires NRC2, NRC3, and NRC4 in a genetically redundant manner to confer resistance to Potato virus X (PVX). Given that some helper NLRs are points of convergence in downstream signaling of multiple upstream sensor NLRs, they would be ideal targets for parasite effectors to overcome NLR-mediated ETI.

Delivery of gene editing components such as the Cas nuclease and single guide RNAs (sgRNAs) into plant cells is commonly accomplished by agrobacterium-mediated transformation. Although Arabidopsis is easy to transform, generation of biallelic edited plants requires screening a large number of plants in subsequent generations. Here, we describe optimization of the Tobacco rattle virus (TRV) for in planta delivery of sgRNAs fused to a tRNAIleu that induces efficient multiplex somatic and biallelic heritable editing in Arabidopsis. Inclusion of tRNAIleu enhances the systemic movement of TRV and the mutant phenotype is visible in the initial TRV::sgRNA-tRNAIleu infected Arabidopsis, which allows for the uncovering of lethal phenotypes. Mutant progeny are recovered in the next generation (M1) at frequencies ranging from 30-60%, with 100% mutant recovery in the following (M2) generation. TRV::tRNAIleu system described here allows generation of biallelic edited plants in a single generation and is amenable for large-scale high throughput CRISPR screens.