Prime editing (PE) technology enables precise alterations in the genetic code of a genome of interest. PE offers great potential for identifying major agronomically important genes in plants and editing them into superior variants, ideally targeting multiple loci simultaneously to realize the collective effects of the edits. Here, we report the development of a modular assembly-based multiplex PE system in rice and demonstrate its efficacy in editing up to four genes in a single transformation experiment. The duplex PE (DPE) system achieved a co-editing efficiency of 46.1% in the T0 generation, converting TFIIAγ5 to xa5 and xa23 to Xa23SW11. The resulting double-mutant lines exhibited robust broad-spectrum resistance against multiple Xanthomonas oryzae pathovar oryzae (Xoo) strains in the T1 generation. In addition, we successfully edited OsEPSPS1 to an herbicide-tolerant variant and OsSWEET11a to a Xoo-resistant allele, achieving a co-editing rate of 57.14%. Furthermore, with the quadruple PE (QPE) system, we edited four genes-two for herbicide tolerance (OsEPSPS1 and OsALS1) and two for Xoo resistance (TFIIAγ5 and OsSWEET11a)-using one construct, with a co-editing efficiency of 43.5% for all four genes in the T0 generation. We performed multiplex PE using five more constructs, including two for triplex PE (TPE) and three for QPE, each targeting a different set of genes. The editing rates were dependent on the activity of pegRNA and/or ngRNA. For instance, optimization of ngRNA increased the PE rates for one of the targets (OsSPL13) from 0% to 30% but did not improve editing at another target (OsGS2). Overall, our modular assembly-based system yielded high PE rates and streamlined the cloning of PE reagents, making it feasible for more labs to utilize PE for their editing experiments. These findings have significant implications for advancing gene editing techniques in plants and may pave the way for future agricultural applications.
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High‐efficiency prime editing enables new strategies for broad‐spectrum resistance to bacterial blight of rice
Using genetic resistance against bacterial blight (BB) caused by
- NSF-PAR ID:
- 10443057
- Publisher / Repository:
- Wiley-Blackwell
- Date Published:
- Journal Name:
- Plant Biotechnology Journal
- Volume:
- 21
- Issue:
- 7
- ISSN:
- 1467-7644
- Page Range / eLocation ID:
- p. 1454-1464
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Summary Effective and durable disease resistance for bacterial blight (BB) of rice is a continuous challenge due to the evolution and adaptation of the pathogen,
Xanthomonas oryzae pv.oryzae (Xoo ), on cultivated rice varieties. Fundamental to this pathogens’ virulence is transcription activator‐like (TAL) effectors that activate transcription of host genes and contribute differently to pathogen virulence, fitness or both. Host plant resistance is predicted to be more durable if directed at strategic virulence factors that impact both pathogen virulence and fitness. We characterized Tal7b, a minor‐effect virulence factor that contributes incrementally to pathogen virulence in rice, is a fitness factor to the pathogen and is widely present in geographically diverse strains ofXoo . To identify sources of resistance to this conserved effector, we used a highly virulent strain carrying a plasmid borne copy of Tal7b to screen an indica multi‐parent advanced generation inter‐cross (MAGIC) population. Of 18 QTL revealed by genome‐wide association studies and interval mapping analysis, six were specific to Tal7b (qBB‐tal7b ). Overall, 150 predicted Tal7b gene targets overlapped with qBB‐tal7b QTL. Of these, 21 showed polymorphisms in the predicted effector binding element (EBE) site and 23 lost the EBE sequence altogether. Inoculation and bioinformatics studies suggest that the Tal7b target in one of the Tal7b‐specific QTL, qBB‐tal7b ‐8, is a disease susceptibility gene and that the resistance mechanism for this locus may be through loss of susceptibility. Our work demonstrates that minor‐effect virulence factors significantly contribute to disease and provide a potential new approach to identify effective disease resistance. -
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Abstract Background Xanthomonas oryzae pv.oryzae (Xoo ) causes bacterial leaf blight, a devastating disease of rice. Among the type-3 effectors secreted byXoo to support pathogen virulence, the Transcription Activator-Like Effector (TALE) family plays a critical role. Some TALEs are major virulence factors that activate susceptibility (S ) genes, overexpression of which contributes to disease development. Host incompatibility can result from TALE-induced expression of so-called executor (E ) genes leading to a strong and rapid resistance response that blocks disease development. In that context, the TALE functions as an avirulence (Avr) factor. To date no such avirulence factors have been identified in African strains ofXoo .Results With respect to the importance of TALEs in the Rice-
Xoo pathosystem, we aimed at identifying those that may act as Avr factor within AfricanXoo . We screened 86 rice accessions, and identified 12 that were resistant to two African strains while being susceptible to a well-studied Asian strain. In a gain of function approach based on the introduction of each of the ninetal genes of the avirulent African strain MAI1 into the virulent Asian strain PXO99A, four were found to trigger resistance on specific rice accessions. Loss-of-function mutational analysis further demonstrated theavr activity of two of them,talD andtalI, on the rice varieties IR64 and CT13432 respectively. Further analysis of TalI demonstrated the requirement of its activation domain for triggering resistance in CT13432. Resistance in 9 of the 12 rice accessions that were resistant against AfricanXoo specifically, including CT13432, could be suppressed or largely suppressed by trans-expression of the truncTALEtal2h , similarly to resistance conferred by theXa1 gene which recognizes TALEs generally independently of their activation domain.Conclusion We identified and characterized TalD and TalI as two African
Xoo TALEs with avirulence activity on IR64 and CT13432 respectively. Resistance of CT13432 against AfricanXoo results from the combination of two mechanisms, one relying on the TalI-mediated induction of an unknown executor gene and the other on anXa1 -like gene or allele. -
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Summary SWEETs play important roles in intercellular sugar transport. Induction of SWEET sugar transporters by Transcription Activator‐Like effectors (TALe) of
Xanthomonas ssp. is key for virulence in rice, cassava and cotton.We identified OsSWEET11b with roles in male fertility and potential bacterial blight (BB) susceptibility in rice. While single
ossweet11a or11 b mutants were fertile, double mutants were sterile. As clade III SWEETs can transport gibberellin (GA), a key hormone for spikelet fertility, sterility and BB susceptibility might be explained by GA transport deficiencies. However, in contrast with the Arabidopsis homologues, OsSWEET11b did not mediate detectable GA transport. Fertility and susceptibility therefore are likely to depend on sucrose transport activity.Ectopic induction of
OsSWEET11b by designer TALe enabled TALe‐freeXanthomonas oryzae pv.oryzae (Xoo ) to cause disease, identifyingOsSWEET11b as a potential BB susceptibility gene and demonstrating that the induction of host sucrose uniporter activity is key to virulence ofXoo . Notably, only three of six clade III SWEETs are targeted by knownXoo strains from Asia and Africa.The identification of OsSWEET11b is relevant for fertility and for protecting rice against emerging
Xoo strains that targetOsSWEET11b . -
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Summary Cytosine base editors (CBEs) are great additions to the expanding genome editing toolbox. To improve C‐to‐T base editing in plants, we first compared seven cytidine deaminases in the BE3‐like configuration in rice. We found A3A/Y130F‐CBE_V01 resulted in the highest C‐to‐T base editing efficiency in both rice and
Arabidopsis . Furthermore, we demonstrated this A3A/Y130F cytidine deaminase could be used to improve iSpyMacCas9‐mediated C‐to‐T base editing at A‐rich PAMs. To showcase its applications, we first applied A3A/Y130F‐CBE_V01 for multiplexed editing to generate microRNA‐resistant mRNA transcripts as well as pre‐mature stop codons in multiple seed trait genes. In addition, we harnessed A3A/Y130F‐CBE_V01 for efficient artificial evolution of novelALS andEPSPS alleles which conferred herbicide resistance in rice. To further improve C‐to‐T base editing, multiple CBE_V02, CBE_V03 and CBE_V04 systems were developed and tested in rice protoplasts. The CBE_V04 systems were found to have improved editing activity and purity with focal recruitment of more uracil DNA glycosylase inhibitors (UGIs) by the engineered single guide RNA 2.0 scaffold. Finally, we used whole‐genome sequencing (WGS) to compare six CBE_V01 systems and four CBE_V04 systems for genome‐wide off‐target effects in rice. Different levels of cytidine deaminase‐dependent and sgRNA‐independent off‐target effects were indeed revealed by WGS among edited lines by these CBE systems. We also investigated genome‐wide sgRNA‐dependent off‐target effects by different CBEs in rice. This comprehensive study compared 21 different CBE systems, and benchmarked PmCDA1‐CBE_V04 and A3A/Y130F‐CBE_V04 as next‐generation plant CBEs with high editing efficiency, purity, and specificity.
