CRISPR/Cas-mediated in planta gene targeting: current advances and challenges.

Gene targeting can be used to make modifications at a specific region in a plant's genome and create high-precision tools for plant biotechnology and breeding. However, its low efficiency is a major barrier to its use in plants. The discovery of CRISPR (clustered regularly interspaced short palindromic repeats)-Cas-based site-specific nucleases capable of inducing double-strand breaks in desired loci resulted in the development of novel approaches for plant gene targeting. Several studies have recently demonstrated improvements in gene targeting efficiency through cell-type-specific expression of Cas nucleases, the use of self-amplified gene-targeting-vector DNA, or manipulation of RNA silencing and DNA repair pathways. In this review, we summarize recent advances in CRISPR/Cas-mediated gene targeting in plants and discuss potential efficiency improvements. Increasing the efficiency of gene targeting technology will help pave the way for increased crop yields and food safety in environmentally friendly agriculture.

The immune repressor BIR1 contributes to antiviral defense and undergoes transcriptional and post-transcriptional regulation during viral infections.

BIR1 is a receptor-like kinase that functions as a negative regulator of basal immunity and cell death in Arabidopsis. Using Arabidopsis thaliana and Tobacco rattle virus (TRV), we investigate the antiviral role of BIR1, the molecular mechanisms of BIR1 gene expression regulation during viral infections, and the effects of BIR1 overexpression on plant immunity and development. We found that SA acts as a signal molecule for BIR1 activation during infection. Inactivating mutations of BIR1 in the bir1-1 mutant cause strong antiviral resistance independently of constitutive cell death or SA defense priming. BIR1 overexpression leads to severe developmental defects, cell death and premature death, which correlate with the constitutive activation of plant immune responses. Our findings suggest that BIR1 acts as a negative regulator of antiviral defense in plants, and indicate that RNA silencing contributes, alone or in conjunction with other regulatory mechanisms, to define a threshold expression for proper BIR1 function beyond which an autoimmune response may occur. This work provides novel mechanistic insights into the regulation of BIR1 homeostasis that may be common for other plant immune components.

Tobacco rattle virus 16K silencing suppressor binds ARGONAUTE 4 and inhibits formation of RNA silencing complexes.

The cysteine-rich 16K protein of tobacco rattle virus (TRV), the type member of the genus Tobravirus, is known to suppress RNA silencing. However, the mechanism of action of the 16K suppressor is not well understood. In this study, we used a GFP-based sensor strategy and an Agrobacterium-mediated transient assay in Nicotiana benthamiana to show that 16K was unable to inhibit the activity of existing small interfering RNA (siRNA)- and microRNA (miRNA)-programmed RNA-induced silencing effector complexes (RISCs). In contrast, 16K efficiently interfered with de novo formation of miRNA- and siRNA-guided RISCs, thus preventing cleavage of target RNA. Interestingly, we found that transiently expressed endogenous miR399 and miR172 directed sequence-specific silencing of complementary sequences of viral origin. 16K failed to bind small RNAs, although it interacted with ARGONAUTE 4, as revealed by bimolecular fluorescence complementation and immunoprecipitation assays. Site-directed mutagenesis demonstrated that highly conserved cysteine residues within the N-terminal and central regions of the 16K protein are required for protein stability and/or RNA silencing suppression.

Activation of senescence-associated Dark-inducible (DIN) genes during infection contributes to enhanced susceptibility to plant viruses.

Virus infections in plants cause changes in host gene expression that are common to other environmental stresses. In this work, we found extensive overlap in the transcriptional responses between Arabidopsis thaliana plants infected with Tobacco rattle virus (TRV) and plants undergoing senescence. This is exemplified by the up-regulation during infection of several senescence-associated Dark-inducible (DIN) genes, including AtDIN1 (Senescence 1, SEN1), AtDIN6 (Asparagine synthetase 1, AtASN1) and AtDIN11. DIN1, DIN6 and DIN11 homologues were also activated in Nicotiana benthamiana in response to TRV and Potato virus X (PVX) infection. Reduced TRV levels in RNA interference (RNAi) lines targeting AtDIN11 indicate that DIN11 is an important modulator of susceptibility to TRV in Arabidopsis. Furthermore, low accumulation of TRV in Arabidopsis protoplasts from RNAi lines suggests that AtDIN11 supports virus multiplication in this species. The effect of DIN6 on virus accumulation was negligible in Arabidopsis, perhaps as a result of gene or functional redundancy. However, TRV-induced silencing of NbASN, the DIN6 homologue in N. benthamiana, compromises TRV and PVX accumulation in systemically infected leaves. Interestingly, NbASN inactivation correlates with the appearance of morphological defects in infected leaves. We found that DIN6 and DIN11 regulate virus multiplication in a step prior to the activation of plant defence responses. We hypothesize on the possible roles of DIN6 and DIN11 during virus infection.