Khaoula Belhaj

Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas system

Targeted genome engineering (also known as genome editing) has emerged as an alternative to classical plant breeding and transgenic (GMO) methods to improve crop plants. Until recently, available tools for introducing site-specific double strand DNA breaks were restricted to zinc finger nucleases (ZFNs) and TAL effector nucleases (TALENs). However, these technologies have not been widely adopted by the plant research community due to complicated design and laborious assembly of specific DNA binding proteins for each target gene. Recently, an easier method has emerged based on the bacterial type II CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) immune system. The CRISPR/Cas system allows targeted cleavage of genomic DNA guided by a customizable small noncoding RNA, resulting in gene modifications by both non-homologous end joining (NHEJ) and homology-directed repair (HDR) mechanisms. In this review we summarize and discuss recent applications of the CRISPR/Cas technology in plants.

Editing plant genomes with CRISPR/Cas9

CRISPR/Cas9 is a rapidly developing genome editing technology that has been successfully applied in many organisms, including model and crop plants. Cas9, an RNA-guided DNA endonuclease, can be targeted to specific genomic sequences by engineering a separately encoded guide RNA with which it forms a complex. As only a short RNA sequence must be synthesized to confer recognition of a new target, CRISPR/Cas9 is a relatively cheap and easy to implement technology that has proven to be extremely versatile. Remarkably, in some plant species, homozygous knockout mutants can be produced in a single generation. Together with other sequence-specific nucleases, CRISPR/Cas9 is a game-changing technology that is poised to revolutionise basic research and plant breeding.

Effector Biology of Plant-Associated Organisms: Concepts and Perspectives

Every plant is closely associated with a variety of living organisms. Therefore, deciphering how plants interact with mutualistic and parasitic organisms is essential for a comprehensive understanding of the biology of plants. The field of plant-biotic interactions has recently coalesced around an integrated model. Major classes of molecular players both from plants and their associated organisms have been revealed. These include cell surface and intracellular immune receptors of plants as well as apoplastic and host-cell-translocated (cytoplasmic) effectors of the invading organism. This article focuses on effectors, molecules secreted by plant-associated organisms that alter plant processes. Effectors have emerged as a central class of molecules in our integrated view of plant-microbe interactions. Their study has significantly contributed to advancing our knowledge of plant hormones, plant development, plant receptors, and epigenetics. Many pathogen effectors are extraordinary examples of biological innovation; they include some of the most remarkable proteins known to function inside plant cells. Here, we review some of the key concepts that have emerged from the study of the effectors of plant-associated organisms. In particular, we focus on how effectors function in plant tissues and discuss future perspectives in the field of effector biology.

An effector of the Irish potato famine pathogen antagonizes a host autophagy cargo receptor.

Plants use autophagy to safeguard against infectious diseases. However, how plant pathogens interfere with autophagy-related processes is unknown. Here, we show that PexRD54, an effector from the Irish potato famine pathogen Phytophthora infestans, binds host autophagy protein ATG8CL to stimulate autophagosome formation. PexRD54 depletes the autophagy cargo receptor Joka2 out of ATG8CL complexes and interferes with Joka2s positive effect on pathogen defense. Thus, a plant pathogen effector has evolved to antagonize a host autophagy cargo receptor to counteract host defenses. DOI: http://dx.doi.org/10.7554/eLife.10856.001

Rerouting of Plant Late Endocytic Trafficking Toward a Pathogen Interface

A number of plant pathogenic and symbiotic microbes produce specialized cellular structures that invade host cells where they remain enveloped by host‐derived membranes. The mechanisms underlying the biogenesis and functions of host–microbe interfaces are poorly understood. Here, we show that plant late endocytic trafficking is diverted toward the extrahaustorial membrane (EHM); a host–pathogen interface that develops in plant cells invaded by Irish potato famine pathogen Phytophthora infestans. A late endosome and tonoplast marker protein Rab7 GTPase RabG3c, but not a tonoplast‐localized sucrose transporter, is recruited to the EHM, suggesting specific rerouting of vacuole‐targeted late endosomes to a host–pathogen interface. We revealed the dynamic nature of this process by showing that, upon activation, a cell surface immune receptor traffics toward the haustorial interface. Our work provides insight into the biogenesis of the EHM and reveals dynamic processes that recruit membrane compartments and immune receptors to this host–pathogen interface.

Helper NLR proteins NRC2a/b and NRC3 but not NRC1 are required for Pto‐mediated cell death and resistance in Nicotiana benthamiana

Plants defend against pathogens using both cell surface andintracellular immune receptors (Dodds & Rathjen, 2010; Winet al., 2012). Plant cell surface receptors include receptor-likekinases (RLKs) and receptor-like proteins (RLPs), which respondto pathogen-derived apoplastic molecules (Boller & Felix, 2009;Thomma et al., 2011). By contrast, plant intracellular immunereceptors are typically nucleotide-binding leucine-rich repeat (NB-LRR or NLR) proteins, which respond to translocated effectorsfrom a diversity of pathogens (Eitas & Dangl, 2010; Bonardi et al.,2012). These receptors engage in microbial perce ption either bydirectly binding pathogen molecules or ind irectly by sensingpathogen-induced perturbations (Win et al., 2012). However,signaling events downstream of pathogen recognition remainpoorly understood.In addition to their role in microbial recognition, some NLRproteins contribute to signal transduction and/or amplification(Gabriels et al., 2007; Bonardi et al., 2011; Cesari et al., 2014). Anemerging model is that NLR proteins often function in pairs, with‘helper’ proteins required for the activity of ‘sensors’ that mediatepathogen recognition (Bonardi et al., 2011, 2012). Amongpreviously reported NLR helpers, NRC1 (NB-LRR proteinrequired for hypersensitive-response (HR)-associated cell death 1)stands out for having been reported as a signaling hub required forthe cell death mediated by both cell surface immune receptors suchas Cf-4, Cf-9, Ve1 and LeEix2, as well as intracellular immunereceptors, namely Pto, Rx and Mi-1.2 (Gabriels et al., 2006,2007; Sueldo, 2014; Sueldo et al., 2015). However, these studiesdid not take into account the Nicotiana benthamiana genomesequence, and it remains questionable whether NRC1 is indeedrequired for the reported phenotypes.Functional analyses of NRC1 were performed using virus-induced gene silencing (VIGS) (Gabriels et al., 2007), a method thatis popular for genetic analyses in several plant systems, particularlythe model solanaceous plant N. benthamiana (Burch-Smith et al.,2004). However, interpretation of VIGS can be problematic as theexperiment can result in off-target silencing (Senthil-Kumar & Mysore, 2011). In addition, heterologous gene fragments from other species (e.g. tomato) have been frequently used to silence homologs in N. benthamiana, particularly in studies that predate the sequencing of the N. benthamiana genome (Burton et al., 2000; Liu et al., 2002b; Lee et al., 2003; Gabriels et al., 2006, 2007; SenthilKumaret al., 2007; Oh et al., 2010). In the NRC1 study, a fragment of a tomato gene corresponding to the LRR domain was used for silencing in N. benthamiana (Gabriels et al., 2007). Given that a draft genome sequence of N. benthamiana has been generated (Bombarely et al., 2012) and silencing prediction tools have become available (Fernandez-Pozo et al., 2015), we can now design better VIGS experiments and revisit previously published studies. Two questions arise about the NRC1 study. First, is there a NRC1 ortholog in N. benthamiana? Second, are the reported phenotypes caused by silencing of NRC1 in N. benthamiana? In this study, we investigated NRC1-like genes in solanaceous plants using a combination of genome annotation, phylogenetics, gene silencing and genetic complementation experiments. We discovered three paralogs of NRC1, which we termed NRC2a, NRC2b and NRC3, are required for hypersensitive cell death and resistance mediated by Pto, but are not essential for the cell death triggered by Rx and Mi-1.2. NRC2a/b and NRC3 weakly contribute to the hypersensitive cell death triggered by Cf-4. Our results highlight the importance of applying genetic complementation assays to validate gene function in RNA silencing experiments.

Functional Divergence of Two Secreted Immune Proteases of Tomato

Rcr3 and Pip1 are paralogous secreted papain-like proteases of tomato. Both proteases are inhibited by Avr2 from the fungal pathogen Cladosporium fulvum, but only Rcr3 acts as a co-receptor for Avr2 recognition by the tomato Cf-2 immune receptor. Here, we show that Pip1-depleted tomato plants are hyper-susceptible to fungal, bacterial, and oomycete plant pathogens, demonstrating that Pip1 is an important broad-range immune protease. By contrast, in the absence of Cf-2, Rcr3 depletion does not affect fungal and bacterial infection levels but causes increased susceptibility only to the oomycete pathogen Phytophthora infestans. Rcr3 and Pip1 reside on a genetic locus that evolved over 36 million years ago. These proteins differ in surface-exposed residues outside the substrate-binding groove, and Pip1 is 5- to 10-fold more abundant than Rcr3. We propose a model in which Rcr3 and Pip1 diverged functionally upon gene duplication, possibly driven by an arms race with pathogen-derived inhibitors or by coevolution with the Cf-2 immune receptor detecting inhibitors of Rcr3, but not of Pip1.

NLR network mediates immunity to diverse plant pathogens

Significance Plant and animal nucleotide-binding domain and leucine-rich repeat-containing (NLR) proteins often function in pairs to mediate innate immunity to pathogens. However, the degree to which NLR proteins form signaling networks beyond genetically linked pairs is poorly understood. In this study, we discovered that a large NLR immune signaling network with a complex genetic architecture confers immunity to oomycetes, bacteria, viruses, nematodes, and insects. The network emerged over 100 Mya from a linked NLR pair that diversified into up to one-half of the NLRs of asterid plants. We propose that this NLR network increases robustness of immune signaling to counteract rapidly evolving plant pathogens. Both plants and animals rely on nucleotide-binding domain and leucine-rich repeat-containing (NLR) proteins to respond to invading pathogens and activate immune responses. An emerging concept of NLR function is that “sensor” NLR proteins are paired with “helper” NLRs to mediate immune signaling. However, our fundamental knowledge of sensor/helper NLRs in plants remains limited. In this study, we discovered a complex NLR immune network in which helper NLRs in the NRC (NLR required for cell death) family are functionally redundant but display distinct specificities toward different sensor NLRs that confer immunity to oomycetes, bacteria, viruses, nematodes, and insects. The helper NLR NRC4 is required for the function of several sensor NLRs, including Rpi-blb2, Mi-1.2, and R1, whereas NRC2 and NRC3 are required for the function of the sensor NLR Prf. Interestingly, NRC2, NRC3, and NRC4 redundantly contribute to the immunity mediated by other sensor NLRs, including Rx, Bs2, R8, and Sw5. NRC family and NRC-dependent NLRs are phylogenetically related and cluster into a well-supported superclade. Using extensive phylogenetic analysis, we discovered that the NRC superclade probably emerged over 100 Mya from an NLR pair that diversified to constitute up to one-half of the NLRs of asterids. These findings reveal a complex genetic network of NLRs and point to a link between evolutionary history and the mechanism of immune signaling. We propose that this NLR network increases the robustness of immune signaling to counteract rapidly evolving plant pathogens.

Structural basis of host Autophagy-related protein 8 (ATG8) binding by the Irish potato famine pathogen effector protein PexRD54

Filamentous plant pathogens deliver effector proteins to host cells to promote infection. The Phytophthora infestans RXLR-type effector PexRD54 binds potato ATG8 via its ATG8 family-interacting motif (AIM) and perturbs host-selective autophagy. However, the structural basis of this interaction remains unknown. Here, we define the crystal structure of PexRD54, which includes a modular architecture, including five tandem repeat domains, with the AIM sequence presented at the disordered C terminus. To determine the interface between PexRD54 and ATG8, we solved the crystal structure of potato ATG8CL in complex with a peptide comprising the effectors AIM sequence, and we established a model of the full-length PexRD54-ATG8CL complex using small angle x-ray scattering. Structure-informed deletion of the PexRD54 tandem domains reveals retention of ATG8CL binding in vitro and in planta. This study offers new insights into structure/function relationships of oomycete RXLR effectors and how these proteins engage with host cell targets to promote disease.

A Recent Expansion of the RXLR Effector Gene Avrblb2 Is Maintained in Global Populations of Phytophthora infestans Indicating Different Contributions to Virulence

The introgression of disease resistance (R) genes encoding immunoreceptors with broad-spectrum recognition into cultivated potato appears to be the most promising approach to achieve sustainable management of late blight caused by the oomycete pathogen Phytophthora infestans. Rpi-blb2 from Solanum bulbocastanum shows great potential for use in agriculture based on preliminary potato disease trials. Rpi-blb2 confers immunity by recognizing the P. infestans avirulence effector protein AVRblb2 after it is translocated inside the plant cell. This effector belongs to the RXLR class of effectors and is under strong positive selection. Structure-function analyses revealed a key polymorphic amino acid (position 69) in AVRblb2 effector that is critical for activation of Rpi-blb2. In this study, we reconstructed the evolutionary history of the Avrblb2 gene family and further characterized its genetic structure in worldwide populations. Our data indicate that Avrblb2 evolved as a single-copy gene in a putative ancestral species of P. infestans and has recently expanded in the Phytophthora spp. that infect solanaceous hosts. As a consequence, at least four variants of AVRblb2 arose in P. infestans. One of these variants, with a Phe residue at position 69, evades recognition by the cognate resistance gene. Surprisingly, all Avrblb2 variants are maintained in pathogen populations. This suggests a potential benefit for the pathogen in preserving duplicated versions of AVRblb2, possibly because the variants may have different contributions to pathogen fitness in a diversified solanaceous host environment.