Search for: All records

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. 

Summary Plants have evolved a sophisticated immunity system for specific detection of pathogens and rapid induction of measured defences. Over‐ or constitutive activation of defences would negatively affect plant growth and development. Hence, the plant immune system is under tight positive and negative regulation. MAP kinase phosphatase1 (MKP1) has been identified as a negative regulator of plant immunity in model plantArabidopsis. However, the molecular mechanisms by which MKP1 regulates immune signalling in wheat (Triticum aestivum) are poorly understood. In this study, we investigated the role of TaMKP1 in wheat defence against two devastating fungal pathogens and determined its subcellular localization. We demonstrated that knock‐down ofTaMKP1by CRISPR/Cas9 in wheat resulted in enhanced resistance to rust caused byPuccinia striiformisf. sp.tritici(Pst) and powdery mildew caused byBlumeria graminisf. sp.tritici(Bgt), indicating thatTaMKP1negatively regulates disease resistance in wheat. Unexpectedly, whileTamkp1mutant plants showed increased resistance to the two tested fungal pathogens they also had higher yield compared with wild‐type control plants without infection. Our results suggested that TaMKP1 interacts directly with dephosphorylated and activated TaMPK3/4/6, and TaMPK4 interacts directly with TaPAL. Taken together, we demonstrated TaMKP1 exert negative modulating roles in the activation of TaMPK3/4/6, which are required for MAPK‐mediated defence signalling. This facilitates our understanding of the important roles of MAP kinase phosphatases and MAPK cascades in plant immunity and production, and provides germplasm resources for breeding for high resistance and high yield. 

Abstract Grapevine (Vitis vinifera) is an economically important fruit crop worldwide. The widely cultivated grapevine is susceptible to powdery mildew caused by Erysiphe necator. In this study, we used CRISPR-Cas9 to simultaneously knock out VviWRKY10 and VviWRKY30 encoding two transcription factors reported to be implicated in defense regulation. We generated 53 wrky10 single mutant transgenic plants and 15 wrky10 wrky30 double mutant transgenic plants. In a 2-yr field evaluation of powdery mildew resistance, the wrky10 mutants showed strong resistance, while the wrky10 wrky30 double mutants showed moderate resistance. Further analyses revealed that salicylic acid (SA) and reactive oxygen species contents in the leaves of wrky10 and wrky10 wrky30 were substantially increased, as was the ethylene (ET) content in the leaves of wrky10. The results from dual luciferase reporter assays, electrophoretic mobility shift assays and chromatin immunoprecipitation (ChIP) assays demonstrated that VviWRKY10 could directly bind to the W-boxes in the promoter of SA-related defense genes and inhibit their transcription, supporting its role as a negative regulator of SA-dependent defense. By contrast, VviWRKY30 could directly bind to the W-boxes in the promoter of ET-related defense genes and promote their transcription, playing a positive role in ET production and ET-dependent defense. Moreover, VviWRKY10 and VviWRKY30 can bind to each other's promoters and mutually inhibit each other's transcription. Taken together, our results reveal a complex mechanism of regulation by VviWRKY10 and VviWRKY30 for activation of measured and balanced defense responses against powdery mildew in grapevine. 

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. 

Crop diseases are responsible for substantial yield losses worldwide, thereby threatening global food security. In this Research Topic, a collection of high-quality articles reported recent research progress concerning genes, proteins, secondary metabolites involved in the interactions between crop plants and their pathogens as well as utilization of new synthetic chemicals in control of crop diseases. As co-editors of this research topic, we appreciate the contributions from the authors of the papers published under this topic and highlight the three themes drawn from their research findings. 

Arabidopsis RESISTANCE TO POWDERY MILDEW 8.2 (RPW8.2) is specifically induced by the powdery mildew (PM) fungus (Golovinomyces cichoracearum) in the infected epidermal cells to activate immunity. However, the mechanism of RPW8.2-induction is not well understood. Here, we identify a G. cichoracearum effector that interacts with RPW8.2, named Gc-RPW8.2 interacting protein 1 (GcR8IP1), by a yeast two-hybrid screen of an Arabidopsis cDNA library. GcR8IP1 physically associated with RPW8.2 with its RING finger domain that is essential and sufficient for the association. GcR8IP1 was secreted and translocated into the nucleus of host cell infected with PM. Association of GcR8IP1 with RPW8.2 led to an increase of RPW8.2 in the nucleus. In turn, the nucleus-localised RPW8.2 promoted the activity of the RPW8.2 promoter, resulting in transcriptional self-amplification of RPW8.2 to boost immunity at infection sites. Additionally, ectopic expression or host-induced gene silencing of GcR8IP1 supported its role as a virulence factor in PM. Altogether, our results reveal a mechanism of RPW8.2-dependent defense strengthening via altered partitioning of RPW8.2 and transcriptional self-amplification triggered by a PM fungal effector, which exemplifies an atypical form of effector-triggered immunity. 

Plant subtilases (SBTs) or subtilisin-like proteases comprise a very diverse family of serine peptidases that participates in a broad spectrum of biological functions. Despite increasing evidence for roles of SBTs in plant immunity in recent years, little is known about wheat (Triticum aestivum) SBTs (TaSBTs). Here, we identified 255 TaSBT genes from bread wheat using the latest version 2.0 of the reference genome sequence. The SBT family can be grouped into five clades, from TaSBT1 to TaSBT5, based on a phylogenetic tree constructed with deduced protein sequences. In silico protein-domain analysis revealed the existence of considerable sequence diversification of the TaSBT family which, together with the local clustered gene distribution, suggests that TaSBT genes have undergone extensive functional diversification. Among those TaSBT genes whose expression was altered by biotic factors, TaSBT1.7 was found to be induced in wheat leaves by chitin and flg22 elicitors, as well as six examined pathogens, implying a role for TaSBT1.7 in plant defense. Transient overexpression of TaSBT1.7 in Nicotiana benthamiana leaves resulted in necrotic cell death. Moreover, knocking down TaSBT1.7 in wheat using barley stripe mosaic virus-induced gene silencing compromised the hypersensitive response and resistance against Puccinia striiformis f. sp. tritici, the causal agent of wheat stripe rust. Taken together, this study defined the full complement of wheat SBT genes and provided evidence for a positive role of one particular member, TaSBT1.7, in the incompatible interaction between wheat and a stripe rust pathogen.