BackgroundThe CRISPR/Cas9 system provides bacteria and archaea with molecular immunity against invading phages and conjugative plasmids. Recently, CRISPR/Cas9 has been used for targeted genome editing in diverse eukaryotic species.ResultsIn this study, we investigate whether the CRISPR/Cas9 system could be used in plants to confer molecular immunity against DNA viruses. We deliver sgRNAs specific for coding and non-coding sequences of tomato yellow leaf curl virus (TYLCV) into Nicotiana benthamiana plants stably overexpressing the Cas9 endonuclease, and subsequently challenge these plants with TYLCV. Our data demonstrate that the CRISPR/Cas9 system targeted TYLCV for degradation and introduced mutations at the target sequences. All tested sgRNAs exhibit interference activity, but those targeting the stem-loop sequence within the TYLCV origin of replication in the intergenic region (IR) are the most effective. N. benthamiana plants expressing CRISPR/Cas9 exhibit delayed or reduced accumulation of viral DNA, abolishing or significantly attenuating symptoms of infection. Moreover, this system could simultaneously target multiple DNA viruses.ConclusionsThese data establish the efficacy of the CRISPR/Cas9 system for viral interference in plants, thereby extending the utility of this technology and opening the possibility of producing plants resistant to multiple viral infections.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0799-6) contains supplementary material, which is available to authorized users.

Targeted genome editing in plants will not only facilitate functional genomics studies but also help to discover, expand, and create novel traits of agricultural importance (Pennisi, 2010). The most widely used approach for editing plant genomes involves generating targeted double-strand DNA breaks (DSBs) and harnessing the two main DSB repair pathways: imprecise non-homologous end joining and precise homology-directed repair (Voytas, 2013). Enzymes that specifically bind the userselected genomic sequences to create DSBs can be generated de novo as synthetic bimodular proteins containing a DNAbinding module, engineered to bind a user-defined sequence, along with a DNA-cleaving module, capable of making DSBs. Several classes of nucleases have been developed with DNAbinding domains engineered to confer sequence specificity, including homing endonucleases, zinc finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs). Customization of these genome editing platforms, however, requires protein engineering, a time-consuming and laborintensive process (Puchta and Fauser, 2014). Furthermore, delivery of genome engineering reagents into plant cells is a major barrier to the effective use of these technologies for creating novel traits (Baltes et al., 2014).

SummaryTargeted genomic regulation is a powerful approach to accelerate trait discovery and development in agricultural biotechnology. Bacteria and archaea use clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) regulatory systems for adaptive molecular immunity against foreign nucleic acids introduced by invading phages and conjugative plasmids. The type II CRISPR/Cas system has been adapted for genome editing in many cell types and organisms. A recent study used the catalytically inactive Cas9 (dCas9) protein combined with guide-RNAs (gRNAs) as a DNA-targeting platform to modulate gene expression in bacterial, yeast, and human cells. Here, we modified this DNA-targeting platform for targeted transcriptional regulation in planta by developing chimeric dCas9-based transcriptional activators and repressors. To generate transcriptional activators, we fused the dCas9 Cterminus with the activation domains of EDLL and TAL effectors. To generate a transcriptional repressor, we fused the dCas9 C-terminus with the SRDX repression domain. Our data demonstrate that dCas9 fusion with the EDLL activation domain (dCas9:EDLL) and the TAL activation domain (dCas9:TAD), guided by gRNAs complementary to selected promoter elements, induce strong transcriptional activation on Bs3::uidA targets in plant cells. Further, the dCas9:SRDX-mediated transcriptional repression of an endogenous gene. Thus, our results suggest that the synthetic transcriptional repressor (dCas9:SRDX) and activators (dCas9:EDLL and dCas9:TAD) can be used as endogenous transcription factors to repress or activate transcription of an endogenous genomic target. Our data indicate that the CRISPR/dCas9 DNA-targeting platform can be used in plants as a functional genomics tool and for biotechnological applications.

Pathogen recognition by plants results in the activation of signaling pathways that induce defense reactions. There is growing evidence indicating that epigenetic mechanisms directly participate in plant immune memory. Here, we discuss current knowledge of diverse epigenomic processes and elements, such as noncoding RNAs, DNA and RNA methylation, histone post-translational modifications, and chromatin remodeling, that have been associated with the regulation of immune responses in plants. Furthermore, we discuss the currently limited evidence of transgenerational inheritance of pathogen-induced defense priming, together with its potentials, challenges, and limitations for crop improvement and biotechnological applications.

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In many eukaryotic systems during immune responses, mitogen-activated protein kinases (MAPKs) link cytoplasmic signaling to chromatin events by targeting transcription factors, chromatin remodeling complexes, and the RNA polymerase machinery. So far, knowledge on these events is scarce in plants and no attempts have been made to focus on phosphorylation events of chromatin-associated proteins. Here we carried out chromatin phosphoproteomics upon elicitor-induced activation of Arabidopsis. The events in WT were compared with those in mpk3, mpk4, and mpk6 mutant plants to decipher specific MAPK targets. Our study highlights distinct signaling networks involving MPK3, MPK4, and MPK6 in chromatin organization and modification, as well as in RNA transcription and processing. Among the chromatin targets, we characterized the AT-hook motif containing nuclear localized (AHL) DNA-binding protein AHL13 as a substrate of immune MAPKs. AHL13 knockout mutant plants are compromised in pathogen-associated molecular pattern (PAMP)-induced reactive oxygen species production, expression of defense genes, and PAMP-triggered immunity. Transcriptome analysis revealed that AHL13 regulates key factors of jasmonic acid biosynthesis and signaling and affects immunity toward Pseudomonas syringae and Botrytis cinerea pathogens. Mutational analysis of the phosphorylation sites of AHL13 demonstrated that phosphorylation regulates AHL13 protein stability and thereby its immune functions.

Plant growth regulators have an important role in various developmental processes during the life cycle of plants. They are involved in abiotic stress responses and tolerance. They have very well-developed capabilities to sense the changes in their external milieu and initiate an appropriate signaling cascade that leads to the activation of plant defense mechanisms. The plant defense system activation causes build-up of plant defense hormones like jasmonic acid (JA) and antioxidant systems like glutathione (GSH). Moreover, calcium (Ca2+) transients are also seen during abiotic stress conditions depicting the role of Ca2+ in alleviating abiotic stress as well. Therefore, these growth regulators tend to control plant growth under varying abiotic stresses by regulating its oxidative defense and detoxification system. This review highlights the role of Jasmonates, Calcium, and glutathione in abiotic stress tolerance and activation of possible novel interlinked signaling cascade between them. Further, phyto-hormone crosstalk with jasmonates, calcium and glutathione under abiotic stress conditions followed by brief insights on omics approaches is also elucidated.

Arabidopsis homolog of human G3BP1 negatively regulates plant immunity and defense responses. Atg3bp1 mutant lines show constitutive stomata closure, expression of a number of key defense marker genes, and accumulate salicylic acid.