The 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. In 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. These 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.

Summary: Targeted 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 C‐terminus 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.

Plant trait engineering requires efficient targeted genome-editing technologies. Clustered regularly interspaced palindromic repeats (CRISPRs)/ CRISPR associated (Cas) type II system is used for targeted genome-editing applications across eukaryotic species including plants. Delivery of genome engineering reagents and recovery of mutants remain challenging tasks for in planta applications. Recently, we reported the development of Tobacco rattle virus (TRV)-mediated genome editing in Nicotiana benthamiana. TRV infects the growing points and possesses small genome size; which facilitate cloning, multiplexing, and agroinfections. Here, we report on the persistent activity and specificity of the TRV-mediated CRISPR/Cas9 system for targeted modification of the Nicotiana benthamiana genome. Our data reveal the persistence of the TRV- mediated Cas9 activity for up to 30 d post-agroinefection. Further, our data indicate that TRV-mediated genome editing exhibited no off-target activities at potential off-targets indicating the precision of the system for plant genome engineering. Taken together, our data establish the feasibility and exciting possibilities of using virus-mediated CRISPR/Cas9 for targeted engineering of plant genomes.

The effects of three rhizobacterial isolates namely Pseudomonas fluorescens (M1), Pseudomonas putida (M2) and Bacillus subtilis (M3) were examined to enhance growth and chemical components such as chlorophyll and proline of three cultivars of soybean (Glycine max L.) under two levels of salinity stress (S1 = 200 mM and S2 = 400 mM of NaCl salt). Several morphological and physiological parameters were investigated. The highest mean values of final germination percent (FGP) were registered in cultivar Crawford (95%) followed by Giza111 cultivar (93%) in the presence of P. fluorescens, while, FGP of Clark was 85%. Mean germination time was decreased by the application of P. fluorescens or P. putida in both salt stressed and unstressed traits. All growth parameters were significantly decreased by salinity treatments, particularly at S2. A significant increase in stem length and shoot fresh weight was recorded in plants treated with P. fluorescens. This enhancing trend was followed by the application of P. putida then B. subtilis. Chlorophyll contents and plant soluble proteins were decreased, while proline content was increased as compared with control treatment. Results showed that the salt tolerant cultivar, Crawford, may have a better tolerance strategy against oxidative damages by increasing antioxidant enzymes activities under high salinity stress. These results suggest that salt induced oxidative stress in soybean is generally counteracted by enzymatic defense systems stimulated under harsh conditions. Our results showed that inoculation with plant growth-promoting rhizobacterial (PGPR) alleviated the harmful effects of salinity stress on soybean cultivars. The diversity in the phylogenetic relationship and in the level of genetic among cultivars was assessed by SDS-PAGE and RAPD markers. Among the polymorphism bands, only few were found to be useful as positive or negative markers associated with salt stress. The maximum number of bands (17) was recorded in Crawford, while the minimum number of bands (11) was recorded in Clark. Therefore, the ISSR can be used to identify alleles associated with the salt stress in soybean germplasm.