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1. TAL Effectors with Avirulence Activity in African Strains of Xanthomonas oryzae pv. oryzae

   https://doi.org/10.1186/s12284-022-00553-9  

   Lachaux, Marlène ; Thomas, Emilie ; Bogdanove, Adam J. ; Szurek, Boris ; Hutin, Mathilde ( February 2022 , Rice)

   Xanthomonas oryzaepv.oryzae(Xoo) causes bacterial leaf blight, a devastating disease of rice. Among the type-3 effectors secreted byXooto 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.

   With respect to the importance of TALEs in the Rice-Xoopathosystem, 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 ninetalgenes of the avirulent African strain MAI1 into the virulent Asian strain PXO99^A, four were found to trigger resistance on specific rice accessions. Loss-of-function mutational analysis further demonstrated theavractivity of two of them,talDandtalI,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 AfricanXoospecifically, including CT13432, could be suppressed or largely suppressed by trans-expression of the truncTALEtal2h, similarly to resistance conferred by theXa1gene which recognizes TALEs generally independently of their activation domain.

   We identified and characterized TalD and TalI as two AfricanXooTALEs with avirulence activity on IR64 and CT13432 respectively. Resistance of CT13432 against AfricanXooresults 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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2. High‐efficiency prime editing enables new strategies for broad‐spectrum resistance to bacterial blight of rice

   https://doi.org/10.1111/pbi.14049  

   Gupta, Ajay ; Liu, Bo ; Chen, Qi‐Jun ; Yang, Bing ( May 2023 , Plant Biotechnology Journal)

   Using genetic resistance against bacterial blight (BB) caused byXanthomonas oryzaepathovaroryzae(Xoo) is a major objective in rice breeding programmes. Prime editing (PE) has the potential to create novel germplasm againstXoo. Here, we use an improved prime‐editing system to implement two new strategies for BB resistance. Knock‐in of TAL effector binding elements (EBE) derived from the BB susceptible geneSWEET14into the promoter of a dysfunctional executorRgenexa23reaches 47.2% with desired edits including biallelic editing at 18% in T₀generation that enables an inducible TALE‐dependent BB resistance. Editing the transcription factor TFIIA geneTFIIAγ5required for TAL effector‐dependent BB susceptibility recapitulates the resistance ofxa5at an editing efficiency of 88.5% with biallelic editing rate of 30% in T₀generation. The engineered loci provided resistance against multipleXoostrains in T₁generation. Whole‐genome sequencing detected noOsMLH1dn‐associated random mutations and no off‐target editing demonstrating high specificity of this PE system. This is the first‐ever report to use PE system to engineer resistance against biotic stress and to demonstrate knock‐in of 30‐nucleotides cis‐regulatory element at high efficiency. The new strategies hold promises to fend rice off the evolvingXoostrains and protect it from epidemics.

    

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3. Resistance and susceptibility QTL identified in a rice MAGIC population by screening with a minor‐effect virulence factor from Xanthomonas oryzae pv. oryzae

   https://doi.org/10.1111/pbi.13438  

   Huerta, Alejandra I. ; Delorean, Emily E. ; Bossa‐Castro, Ana M. ; Tonnessen, Bradley W. ; Raghavan, Chitra ; Corral, Rene ; Pérez‐Quintero, Álvaro L. ; Leung, Hei ; Verdier, Valérie ; Leach, Jan E. ( July 2020 , Plant Biotechnology Journal)

   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 oryzaepv.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‐tal7bQTL. 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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4. Natural variation in the expression and catalytic activity of a naringenin 7‐ O ‐methyltransferase influences antifungal defenses in diverse rice cultivars

   https://doi.org/10.1111/tpj.14577  

   Murata, Koichi ; Kitano, Takashige ; Yoshimoto, Riko ; Takata, Ryo ; Ube, Naoki ; Ueno, Kotomi ; Ueno, Makoto ; Yabuta, Yukinori ; Teraishi, Masayoshi ; Holland, Cynthia K. ; et al ( November 2019 , The Plant Journal)

   Phytoalexins play a pivotal role in plant–pathogen interactions. Whereas leaves of rice (Oryza sativa) cultivar Nipponbare predominantly accumulated the phytoalexin sakuranetin after jasmonic acid induction, only very low amounts accumulated in the Kasalath cultivar. Sakuranetin is synthesized from naringenin by naringenin 7‐O‐methyltransferase (NOMT). Analysis of chromosome segment substitution lines and backcrossed inbred lines suggested thatNOMTis the underlying cause of differential phytoalexin accumulation between Nipponbare and Kasalath. Indeed, bothNOMTexpression and NOMT enzymatic activity are lower in Kasalath than in Nipponbare. We identified a proline to threonine substitution in Kasalath relative to Nipponbare NOMT as the main cause of the lower enzymatic activity. Expanding this analysis to rice cultivars with varying amounts of sakuranetin collected from around the world showed thatNOMTinduction is correlated with sakuranetin accumulation. In bioassays withPyricularia oryzae,Gibberella fujikuroi,Bipolaris oryzae,Burkholderia glumae,Xanthomonas oryzae,Erwinia chrysanthemi,Pseudomonas syringae, andAcidovorax avenae, naringenin was more effective against bacterial pathogens and sakuranetin was more effective against fungal pathogens. Therefore, the relative amounts of naringenin and sakuranetin may provide protection against specific pathogen profiles in different rice‐growing environments. In a dendrogram ofNOMTgenes, those from low‐sakuranetin‐accumulating cultivars formed at least two clusters, only one of which involves the proline to threonine mutation, suggesting that the low sakuranetin chemotype was acquired more than once in cultivated rice. Strains of the wild rice speciesOryza rufipogonalso exhibited differential sakuranetin accumulation, indicating that this metabolic diversity predates rice domestication.

    

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5. Modularly assembled multiplex prime editors for simultaneous editing of agronomically important genes in rice

   https://doi.org/10.1016/j.xplc.2023.100741  

   Gupta, Ajay ; Liu, Bo ; Raza, Saad ; Chen, Qi-Jun ; Yang, Bing ( October 2023 , Plant Communications)

   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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