Search for: All records

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. 

Abstract Clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease (Cas) technologies facilitate routine genome engineering of one or a few genes at a time. However, large-scale CRISPR screens with guide RNA libraries remain challenging in plants. Here, we have developed a comprehensive all-in-one CRISPR toolbox for Cas9-based genome editing, cytosine base editing, adenine base editing (ABE), Cas12a-based genome editing and ABE, and CRISPR-Act3.0-based gene activation in both monocot and dicot plants. We evaluated all-in-one T-DNA expression vectors in rice (Oryza sativa, monocot) and tomato (Solanum lycopersicum, dicot) protoplasts, demonstrating their broad and reliable applicability. To showcase the applications of these vectors in CRISPR screens, we constructed guide RNA (gRNA) pools for testing in rice protoplasts, establishing a high-throughput approach to select high-activity gRNAs. Additionally, we demonstrated the efficacy of sgRNA library screening for targeted mutagenesis of ACETOLACTATE SYNTHASE in rice, recovering novel candidate alleles for herbicide resistance. Furthermore, we carried out a CRISPR activation screen in Arabidopsis thaliana, rapidly identifying potent gRNAs for FLOWERING LOCUS T activation that confer an early-flowering phenotype. This toolbox contains 61 versatile all-in-one vectors encompassing nearly all commonly used CRISPR technologies. It will facilitate large-scale genetic screens for loss-of-function or gain-of-function studies, presenting numerous promising applications in plants. 

Abstract In Arabidopsis thaliana, the POWDERY MILDEW RESISTANT4 (PMR4)/GLUCAN SYNTHASE LIKE5 (GSL5) callose synthase is required for pathogen-induced callose deposition in cell wall defense. Paradoxically, pmr4/gsl5 mutants exhibit strong resistance to both powdery and downy mildew. The powdery mildew resistance of pmr4/gsl5 has been attributed to upregulated salicylic acid (SA) signaling based on its dependance on PHYTOALEXIN DEFICIENT4 (PAD4), which controls SA accumulation, and its abolishment by bacterial NahG salicylate hydroxylase. Our study revealed that disruption of PMR4/GSL5 also leads to early senescence. Suppressor analysis uncovered that PAD4 and N-hydroxypipecolic acid (NHP) biosynthetic genes ABERRANT GROWTH AND DEATH2-LIKE DEFENSE RESPONSE PROTEIN1 (ALD1) and FLAVIN-DEPENDENT MONOXYGENASE1 (FMO1) are required for early senescence of pmr4/gsl5 mutants. The critical role of NHP in the early senescence of pmr4/gsl5 was supported by greatly increased accumulation of pipecolic acid in pmr4/gsl5 mutants. In contrast, disruption of the SA biosynthetic gene ISOCHORISMATE SYNTHASE1/SA-INDUCTION DIFFICIENT 2 (ICS1/SID2), which greatly reduces SA accumulation, had little effect on impaired growth of pmr4/gsl5. Furthermore, while disruption of PAD4 completely abolished the powdery mildew resistance in pmr4/gsl5, mutations in ICS1/SID2, ALD1, or FMO1 had only a minor effect on the resistance of the mutant plants. However, disruption of both ICS1/SID2 and FMO1 abolished the enhanced immunity of the callose synthase mutants against the fungal pathogen. Therefore, while NHP plays a crucial role in the early senescence of pmr4/gsl5 mutants, both SA and NHP have important roles in the strong powdery mildew resistance induced by the loss of the callose synthase. 

Summary CRISPR‐Cas‐based cytosine base editors (CBEs) are prominent tools that perform site‐specific and precise C‐to‐T conversions catalysed by cytidine deaminases. However, their use is often constrained by stringent editing preferences for genomic contexts, off‐target effects and restricted editing windows. To expand the repertoire of CBEs, we systematically screened 66 novel cytidine deaminases sourced from various organisms, predominantly from the animal kingdom and benchmarked them in rice protoplasts using the nCas9‐BE3 configuration. After selecting candidates in rice protoplasts and further validation in transgenic rice lines, we unveiled a few cytidine deaminases exhibiting high editing efficiencies and wide editing windows. CBEs based on these cytidine deaminases also displayed minimal frequencies of indels and C‐to‐R (R = A/G) conversions, suggesting high purity in C‐to‐T base editing. Furthermore, we highlight the highly efficient cytidine deaminase OoA3GX2 derived from Orca (killer whale) for its comparable activity across GC/CC/TC/AC sites, thus broadening the targeting scope of CBEs for robust multiplexed base editing. Finally, the whole‐genome sequencing analyses revealed very few sgRNA‐dependent and ‐independent off‐target effects in independent T₀lines. This study expands the cytosine base‐editing toolkit with many cytidine deaminases sourced from mammals, providing better‐performing CBEs that can be further leveraged for sophisticated genome engineering strategies in rice and likely in other plant species. 

Abstract Powdery mildew fungi are obligate biotrophic pathogens that only invade plant epidermal cells. There are two epidermal surfaces in every plant leaf: the adaxial (upper) side and the abaxial (lower) side. While both leaf surfaces can be susceptible to adapted powdery mildew fungi in many plant species, there have been observations of leaf abaxial immunity in some plant species including Arabidopsis. The genetic basis of such leaf abaxial immunity remains unknown. In this study, we tested a series of Arabidopsis mutants defective in one or more known defense pathways with the adapted powdery mildew isolate Golovinomyces cichoracearum UCSC1. We found that leaf abaxial immunity was significantly compromised in mutants impaired for both the EDS1/PAD4- and PEN2/PEN3-dependent defenses. Consistently, expression of EDS1–yellow fluorescent protein and PEN2–green fluorescent protein fusions from their respective native promoters in the respective eds1-2 and pen2-1 mutant backgrounds was higher in the abaxial epidermal cells than in the adaxial epidermal cells. Altogether, our results indicate that leaf abaxial immunity against powdery mildew in Arabidopsis is at least partially due to enhanced EDS1/PAD4- and PEN2/PEN3-dependent defenses. Such transcriptionally pre-programmed defense mechanisms may underlie leaf abaxial immunity in other plant species such as hemp and may be exploited for engineering adaxial immunity against powdery mildew fungi in crop plants. 

Abstract Nonhost resistance (NHR) refers to the immunity of most tested genotypes of a plant species to most tested variants of a pathogen species. Thus, NHR is broad spectrum and durable in nature and constitutes a major safety barrier against invasion of a myriad of potentially pathogenic microbes in any plants including domesticated crops. Genetic study of NHR is generally more difficult compared to host resistance mainly because NHR is genetically more complicated and often lacks intraspecific polymorphisms. Nevertheless, substantial progress has been made towards the understanding of the molecular basis of NHR in the past two decades using various approaches. Not surprisingly, molecular mechanisms of NHR revealed so far encompasses pathogen‐associated molecular pattern‐triggered immunity and effector‐triggered immunity. In this review, we briefly discuss the inherent difficulty in genetic studies of NHR and summarize the main approaches that have been taken to identify genes contributing to NHR. We also discuss new enabling strategies for dissecting multilayered NHR in model plants with a focus on NHR against filamentous pathogens, especially biotrophic pathogens such as powdery mildew and rust fungi. 

Biolistic delivery of intron-optimized LbCas12a and its variants into shoot apical meristems results in efficient multiplexed genome editing in common wheat. 

Recessive mutations in the mildew locus O (MLO) gene were first identified as key factors conferring broad-spectrum resistance to powdery mildew in barley. This discovery inspired extensive research on MLOs and novel breeding strategies for powdery mildew resistance by targetingMLOgenes in various crops. Over the past two decades, studies have revealed broader roles for MLOs beyond powdery mildew susceptibility, including regulating interactions with diverse pathogens and symbionts, root thigmomorphogenesis, and reproductive development. Recent findings identify MLOs as calcium channels, offering a unifying molecular framework for understanding their diverse biological functions. However, significant challenges remain in comprehensively understanding the cellular and molecular mechanisms underlying MLO functions. In this review, we examine the MLO-related literature to delineate the multifaceted roles of MLOs in plant immunity and development. By integrating published phylogenetic, genetic, biochemical, and molecular studies with original in silico analyses, we propose mechanistic models to contextualize the diverse functions of MLOs with a focus on plant immunity and susceptibility.