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Abstract Key messageBiolistic particle bombardment was used to deliver CRISPR-Cas9 ribonucleoprotein complexes (RNP) into the shoot apical meristem tissue of citrus and axillary meristem tissue of poplar, generating directed mutations in target genes. AbstractThe use of meristematic tissues offers a strategic approach to genome editing in woody species, especially those that are recalcitrant to conventional tissue culture, as these regions contain totipotent, highly regenerative cells capable of giving rise to whole plants. Here, we employed biolistic delivery of genome-editing reagents into theshoot apical meristem (SAM) of citrus and the axillary meristems (AXM) of poplar. The system was first validated using a GFP expression construct and subsequently applied for targeted genome editing. In citrus, edited plants were obtained at the CsNPR3 locus exclusively through the delivery of CRISPR/Cas9 ribonucleoproteins (RNPs), whereas plasmid-based vectors were unsuccessful. Similarly, genome editing in poplar was achieved using RNPs targeting the Pt4CL1 gene. Although chimeric events were detected, this approach provides a feasible and innovative framework for producing transgene-free edited perennial plants. 

Abstract Key messageTransgene-free genome editing of the gene of interest in citrus and poplar has been achieved by co-editing theALSgene via transient transgene expression of an efficient cytosine base editor. AbstractCRISPR-Cas genome editing systems have been widely used in plants. However, such genome-edited plants are nearly always transgenic in the first generation whenAgrobacterium-mediated transformation is used. Transgene-free genome-edited plants are valuable for genetic analysis and breeding as well as simplifying regulatory approval. It can be challenging to generate transgene-free genome-edited plants in vegetatively propagated or perennial plants. To advance transgene-free genome editing in citrus and poplar, we investigated a co-editing strategy using an efficient cytosine base editor (CBE) to edit theALSgene to confer herbicide resistance combined with transient transgene expression and potential mobile RNA-based movement of CBE transcripts to neighboring, non-transgenic cells. An FCY-UPP based cytotoxin system was used to select non-transgenic plants that survive after culturing on 5-FC containing medium. While the editing efficiency is higher in poplar than in citrus, our results show that the CBE-based co-editing strategy works in both citrus and poplar, albeit with low efficiency for biallelic edits. Unexpectedly, the addition of the TLS mobile RNA sequence reduced genome editing efficiency in both transgenic and non-transgenic plants. Although a small fraction of escaping plants is detected in both positive and negative selection processes, our data demonstrate a promising approach for generating transgene-free base-edited plants. 

For plant viruses, the ability to load into the vascular phloem and spread systemically within a host is an essential step in establishing a successful infection. However, access to the vascular phloem is highly regulated, representing a significant obstacle to virus loading, movement, and subsequent unloading into distal uninfected tissues. Recent studies indicate that during virus infection, phloem tissues are a source of significant transcriptional and translational alterations, with the number of virus-induced differentially expressed genes being four- to sixfold greater in phloem tissues than in surrounding nonphloem tissues. In addition, viruses target phloem-specific components as a means to promote their own systemic movement and disrupt host defense processes. Combined, these studies provide evidence that the vascular phloem plays a significant role in the mediation and control of host responses during infection and as such is a site of considerable modulation by the infecting virus. This review outlines the phloem responses and directed reprograming mechanisms that viruses employ to promote their movement through the vasculature.