Disruption of OsPHD1, Encoding a UDP-Glucose Epimerase, Causes JA Accumulation and Enhanced Bacterial Blight Resistance in Rice

Lesion mimic mutants (LMMs) have been widely used in experiments in recent years for studying plant physiological mechanisms underlying programmed cell death (PCD) and defense responses. Here, we identified a lesion mimic mutant, lm212-1, which cloned the causal gene by a map-based cloning strategy, and verified this by complementation. The causal gene, OsPHD1, encodes a UDP-glucose epimerase (UGE), and the OsPHD1 was located in the chloroplast. OsPHD1 was constitutively expressed in all organs, with higher expression in leaves and other green tissues. lm212-1 exhibited decreased chlorophyll content, and the chloroplast structure was destroyed. Histochemistry results indicated that H2O2 is highly accumulated and cell death is occurred around the lesions in lm212-1. Compared to the wild type, expression levels of defense-related genes were up-regulated, and resistance to bacterial pathogens Xanthomonas oryzae pv. oryzae (Xoo) was enhanced, indicating that the defense response was activated in lm212-1, ROS production was induced by flg22, and chitin treatment also showed the same result. Jasmonic acid (JA) and methyl jasmonate (MeJA) increased, and the JA signaling pathways appeared to be disordered in lm212-1. Additionally, the overexpression lines showed the same phenotype as the wild type. Overall, our findings demonstrate that OsPHD1 is involved in the regulation of PCD and defense response in rice.

GoldBricks: An Improved Cloning Strategy that Combines Features of Golden Gate and Biobricks for Better Efficiency and Usability

ABSTRACT With increasing complexity of expression studies and the repertoire of characterized sequences, combinatorial cloning has become a common necessity. Techniques like Biobricks and Golden Gate aim to standardize and speed up the process of cloning large constructs while enabling sharing of resources. The Biobricks format provides a simplified and flexible approach to endless assembly with a compact library and useful intermediates but is a slow process, joining only two parts in a cycle. Golden Gate improves upon the speed with use of TypeIIS enzymes and joins several parts in a cycle but requires a larger library of parts and logistical inefficiencies scale up significantly in the multigene format. We present here a method that provides improvement over these techniques by combining their features. By using Type IIS enzymes in a format like Biobricks, we have enabled a faster and efficient assembly with reduced scarring, which performs at a similarly fast pace as Golden Gate, but significantly reduces library size and user input. Additionally, this method enables faster assembly of operon-style constructs, a feature requiring extensive workaround in Golden Gate. Our format allows such inclusions resulting in faster and more efficient assembly.

SPL36 encodes a receptor-like protein kinase precursor and regulates programmed cell death and defense responses in rice

The rice (Oryza sativa) spotted leaf 36 (spl36) mutant was identified from an ethyl methanesulfonate–mutagenized Japonica cultivar Yundao population and was previously shown to display a spontaneous cell death phenotype and enhanced resistance to rice bacterial pathogens. Through the analysis of the expression of related genes, we speculate that spl36 is involved in the disease response by up-regulating the expression of defense-related genes. The results of physiological and biochemical experiments indicated that more cell death occurred in the mutant spl36, and the growth and development of the plant were affected. We have isolated SPL36 via a map-based cloning strategy. A single base substitution was detected in spl36, which results in encoding amino acid changes in the SPL36 protein. The predicted SPL36 encodes a receptor-like protein kinase precursor that contains repeated leucine domains and may be involved in stress response of rice. In the salt treatment experiment, we found that the mutant spl36 showed sensitivity to salt. Therefore, SPL36 may negatively regulate salt stress-related responses.

Reversed paired-gRNA plasmid cloning strategy for efficient genome editing in Escherichia coli.

Background: Co-expression of two distinct guide RNAs (gRNAs) has been used to facilitate the application of CRISPR/Cas9 system in fields such as large genomic deletion. The paired gRNAs are often placed adjacently in the same direction and expressed individually by two identical promoters, constituting direct repeats (DRs) which are susceptible to self-homologous recombination. As a result, the paired-gRNA plasmids cannot remain stable, which greatly prevents scalable application of the CRISPR/Cas9 system. Results: To address this limitation, different DRs-involved paired-gRNA plasmids were designed and the events of recombination were characterized. Deletion between DRs occurred with high frequencies during plasmid construction and subsequent plasmid propagation. This recombination event was RecA-independent, which agreed with the replication slippage model. To increase plasmid stability, a reversed paired-gRNA plasmids (RPGPs) cloning strategy was developed by converting DRs to the more stable invert repeats (IRs), which completely eliminated DRs-induced recombination. Using RPGPs, rapid deletion of chromosome fragments up to 100 kb with an efficiency of 83.33% was achieved in Escherichia coli. Conclusions: The RPGPs cloning strategy serves as a general solution to avoid plasmid RecA-independent recombination. It can be adapted to applications that rely on paired gRNAs or repeated genetic parts.

Reversed paired-gRNA plasmid cloning strategy for efficient genome editing in Escherichia coli

Background: Co-expression of two distinct guide RNAs (gRNAs) has been used to facilitate the application of CRISPR/Cas9 system in fields such as large genomic deletion. The paired gRNAs are often placed adjacently in the same direction and expressed individually by two identical promoters, constituting direct repeats (DRs) which are susceptible to self-homologous recombination. As a result, the paired-gRNA plasmids cannot remain stable, which greatly prevents scalable application of the CRISPR/Cas9 system. Results: To address this limitation, different DRs-involved paired-gRNA plasmids were designed and the events of recombination were characterized. Deletion between DRs occurred with high frequencies during plasmid construction and subsequent plasmid propagation. This recombination event was RecA-independent, which agreed with the replication slippage model. To increase plasmid stability, a reversed paired-gRNA plasmids (RPGPs) cloning strategy was developed by converting DRs to the more stable invert repeats (IRs), which completely eliminated DRs-induced recombination. Using RPGPs, rapid deletion of chromosome fragments up to 100 kb with an efficiency of 83.33% was achieved in Escherichia coli.Conclusions: The RPGPs cloning strategy serves as a general solution to avoid plasmid RecA-independent recombination. It can be adapted to applications that rely on paired gRNAs or repeated genetic parts.

iFLinkC: an iterative functional linker cloning strategy for the combinatorial assembly and recombination of linker peptides with functional domains

Recent years have witnessed increasing efforts to engineer artificial biological functions through recombination of modular-organized toolboxes of protein scaffolds and parts. A critical, yet frequently neglected aspect concerns the identity of peptide linkers or spacers connecting individual domains which remain poorly understood and challenging to assemble. Addressing these limitations, iFlinkC comprises a highly scalable DNA assembly process that facilitates the combinatorial recombination of functional domains with linkers of varying length and flexibility, thereby overcoming challenges with high GC-content and the repeat nature of linker elements. The capacity of iFLinkC is demonstrated in the construction of synthetic protease switches featuring PDZ-FN3-based affinity clamps and single-chain FKBP12-FRB receptors as allosteric inputs. Library screening experiments demonstrate that linker space is highly plastic as the induction of allosterically regulated protease switches can vary from >150-fold switch-ON to >13-fold switch-OFF solely depending on the identity of the connecting linkers and relative orientation of functional domains. In addition, Pro-rich linkers yield the most potent switches contradicting the conventional use of flexible Gly-Ser linkers. Given the ease and efficiency how functional domains can be readily recombined with any type of linker, iFLinkC is anticipated to be widely applicable to the assembly of any type of fusion protein.

Reversed paired-gRNA plasmid cloning strategy for efficient genome editing in Escherichia coli

BackgroundThe CRISPR-Cas9 system is a powerful tool for genome editing in various organisms. Several of its applications, including the generation of large deletions, require co-expression of two distinct guide RNAs (gRNAs). However, the instability of paired-gRNA plasmids prevents these applications from being scalable in Escherichia coli. Coexpressing paired gRNAs under the driving of independent but identical promoters in the same direction triggers plasmid recombination, due to the presence of direct repeats (DRs). ResultsIn this study, plasmid deletion between DRs occurred with high frequencies during plasmid construction and subsequent duplication processes, when three DRs-involved paired-gRNA plasmids cloning strategies were tested. This recombination phenomenon was RecA-independent, in agreement with the replication slippage model. To completely eliminate the DRs-induced plasmid instability, a reversed paired-gRNA plasmids (RPGPs) cloning strategy was developed by converting DRs to the more stable invert repeats (IRs). ConclusionsUsing RPGPs, we achieved a rapid deletion of chromosome fragments up to 100 kb with high efficiency of 83.33% in Escherichia coli. This study provides general solutions to construct stable plasmids containing short DRs, which can improve the performances of CRISPR systems that rely on paired gRNAs, and also facilitate other applications involving repeated genetic parts.