XA21-mediated resistance to Xanthomonas oryzae pv. oryzae is dose dependent.

The rice receptor kinase XA21 confers broad-spectrum resistance to Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of rice bacterial blight disease. To investigate the relationship between the expression level of XA21 and resulting resistance, we generated independent HA-XA21 transgenic rice lines accumulating the XA21 immune receptor fused with an HA epitope tag. Whole-genome sequence analysis identified the T-DNA insertion sites in sixteen independent T0 events. Through quantification of the HA-XA21 protein and assessment of the resistance to Xoo strain PXO99 in six independent transgenic lines, we observed that XA21-mediated resistance is dose dependent. In contrast, based on the four agronomic traits quantified in these experiments, yield is unlikely to be affected by the expression level of HA-XA21. These findings extend our knowledge of XA21-mediated defense and contribute to the growing number of well-defined genomic landing pads in the rice genome that can be targeted for gene insertion without compromising yield.

Directed evolution rice genes with randomly multiplexed sgRNAs assembly of base editors.

CRISPR-based directed evolution is an effective breeding biotechnology to improve agronomic traits in plants. However, its gene diversification is still limited using individual single guide RNA. We described here a multiplexed orthogonal base editor (MoBE), and a randomly multiplexed sgRNAs assembly strategy to maximize gene diversification. MoBE could induce efficiently orthogonal ABE (<36.6%), CBE (<36.0%), and A&CBE (<37.6%) on different targets, while the sgRNA assembling strategy randomized base editing events on various targets. With respective 130 and 84 targets from each strand of the 34th exon of rice acetyl-coenzyme A carboxylase (OsACC), we observed the target-scaffold combination types up to 27 294 in randomly dual and randomly triple sgRNA libraries. We further performed directed evolution of OsACC using MoBE and randomly dual sgRNA libraries in rice, and obtained single or linked mutations of stronger herbicide resistance. These strategies are useful for in situ directed evolution of functional genes and may accelerate trait improvement in rice.

Promoter editing for the genetic improvement of crops.

Gene expression plays a fundamental role in the regulation of agronomically important traits in crop plants. The genetic manipulation of plant promoters through genome editing has emerged as an effective strategy to create favorable traits in crops by altering the expression pattern of the pertinent genes. Promoter editing can be applied in a directed manner, where nucleotide sequences associated with favorable traits are precisely generated. Alternatively, promoter editing can also be exploited as a random mutagenic approach to generate novel genetic variations within a designated promoter, from which elite alleles are selected based on their phenotypic effects. Pioneering studies have demonstrated the potential of promoter editing in engineering agronomically important traits as well as in mining novel promoter alleles valuable for plant breeding. In this review, we provide an update on the application of promoter editing in crops for increased yield, enhanced tolerance to biotic and abiotic stresses, and improved quality. We also discuss several remaining technical bottlenecks and how this strategy may be better employed for the genetic improvement of crops in the future.

Isolation and Characterization of SPOTTED LEAF42 Encoding a Porphobilinogen Deaminase in Rice.

The formation and development of chloroplasts play a vital role in the breeding of high-yield rice (Oryza sativa L.). Porphobilinogen deaminases (PBGDs) act in the early stage of chlorophyll and heme biosynthesis. However, the role of PBGDs in chloroplast development and chlorophyll production remains elusive in rice. Here, we identified the spotted leaf 42 (spl42) mutant, which exhibited a reddish-brown spotted leaf phenotype. The mutant showed a significantly lower chlorophyll content, abnormal thylakoid morphology, and elevated activities of reactive oxygen species (ROS)-scavenging enzymes. Consistently, multiple genes related to chloroplast development and chlorophyll biosynthesis were significantly down-regulated, whereas many genes involved in leaf senescence, ROS production, and defense responses were upregulated in the spl42 mutant. Map-based cloning revealed that SPL42 encodes a PBGD. A C-to-T base substitution occurred in spl42, resulting in an amino acid change and significantly reduced PBGD enzyme activity. SPL42 targets to the chloroplast and interacts with the multiple organelle RNA editing factors (MORFs) OsMORF8-1 and OsMORF8-2 to affect RNA editing. The identification and characterization of spl42 helps in elucidating the molecular mechanisms associated with chlorophyll synthesis and RNA editing in rice.

Phytochrome B mediates dim-light-reduced insect resistance by promoting the ethylene pathway in rice.

Increasing planting density is one of the most effective ways to improve crop yield. However, one major factor that limits crop planting density is the weakened immunity of plants to pathogens and insects caused by dim light (DL) under shade conditions. The molecular mechanism underlying how DL compromises plant immunity remains unclear. Here, we report that DL reduces rice (Oryza sativa) resistance against brown planthopper (BPH; Nilaparvata lugens) by elevating ethylene (ET) biosynthesis and signaling in a Phytochrome B (OsPHYB)-dependent manner. The DL-reduced BPH resistance is relieved in osphyB mutants, but aggravated in OsPHYB overexpressing plants. Further, we found that DL reduces the nuclear accumulation of OsphyB, thus alleviating Phytochrome Interacting Factor Like14 (OsPIL14) degradation, consequently leading to the up-regulation of 1-Aminocyclopropane-1-Carboxylate Oxidase1 (OsACO1) and an increase in ET levels. In addition, we found that nuclear OsphyB stabilizes Ethylene Insensitive Like2 (OsEIL2) by competitively interacting with EIN3 Binding F-Box Protein (OsEBF1) to enhance ET signaling in rice, which contrasts with previous findings that phyB blocks ET signaling by facilitating Ethylene Insensitive3 (EIN3) degradation in other plant species. Thus, enhanced ET biosynthesis and signaling reduces BPH resistance under DL conditions. Our findings provide insights into the molecular mechanism of the light-regulated ET pathway and host-insect interactions and potential strategies for sustainable insect management.

[Study on the identification of standard and false Gancao by Fourier transform infrared spectroscopy].

Standard Gancao (Glycyrrhiza uralensis Fisch) and false Ciguogancao (Glycyrrhiza pallidiflata Batal) were identified fast, nondestructively by Fourier transform infrared spectroscopy (FTIR) combined with derivative spectra and two-dimensional correlation spectroscopy (2D) in the present article. The result shows that although the two kinds of Gancao belong to one genus, there are some certain differences in their chemical components that are reflected in the IR spectra, but with some similarity and dissimilarity in the IR spectra. The two kinds of Gancao are quite different from each other in second derivative spectra and 2D spectra. Based on the differences reflected in the IR and 2D IR, the standard gancao can be identified from the false easily and clearly. The result also proved that there is a relationship between the IR spectra and the chemical components of the herbs. This method is fast, accurate and nondestructive, and the wastage of sample is less. The fast, accurate property of 2D spectroscopy makes it a powerful and new approach to evaluating medicinal herbs impersonally.