Application of improved GalNAc conjugation in development of cost-effective siRNA therapies targeting cardiovascular diseases.

N-Acetylgalactosamine (GalNAc)-conjugated small interfering RNA (siRNA) therapies have received approval for treating both orphan and prevalent diseases. To improve in vivo efficacy and streamline the chemical synthesis process for efficient and cost-effective manufacturing, we conducted this study to identify better designs of GalNAc-siRNA conjugates for therapeutic development. Here, we present data on redesigned GalNAc-based ligands conjugated with siRNAs against angiopoietin-like 3 (ANGPTL3) and lipoprotein (a) (Lp(a)), two target molecules with the potential to address large unmet medical needs in atherosclerotic cardiovascular diseases. By attaching a novel pyran-derived scaffold to serial monovalent GalNAc units before solid-phase oligonucleotide synthesis, we achieved increased GalNAc-siRNA production efficiency with fewer synthesis steps compared to the standard triantennary GalNAc construct L96. The improved GalNAc-siRNA conjugates demonstrated equivalent or superior in vivo efficacy compared to triantennary GalNAc-conjugated siRNAs.

Arabidopsis membrane protein AMAR1 interaction with type III effector XopAM triggers a hypersensitive response.

The efficient infection of plants by the bacteria Xanthomonas campestris pv. campestris (Xcc) depends on its type III effectors (T3Es). Although the functions of AvrE family T3Es have been reported in some bacteria, the member XopAM in Xcc has not been studied. As XopAM has low sequence similarity to reported AvrE-T3Es and different reports have shown that these T3Es have different targets in hosts, we investigated the functions of XopAM in the Xcc-plant interaction. Deletion of xopAM from Xcc reduced its virulence in cruciferous crops but increased virulence in Arabidopsis (Arabidopsis thaliana) Col-0, indicating that XopAM may perform opposite functions depending on the host species. We further found that XopAM is a lipase that may target the cytomembrane and that this activity might be enhanced by its membrane-targeted protein XOPAM-ACTIVATED RESISTANCE 1 (AMAR1) in Arabidopsis Col-0. The binding of XopAM to AMAR1 induced an intense hypersensitive response that restricted Xcc proliferation. Our results showed that the roles of XopAM in Xcc infection are not the same as those of other AvrE-T3Es, indicating that the functions of this type of T3E have differentiated during long-term bacterium‒host interactions.

Resequencing of global Lotus corniculatus accessions reveals population distribution and genetic loci, associated with cyanogenic glycosides accumulation and growth traits.

BACKGROUND: Lotus corniculatus is a widely distributed perennial legume whose great adaptability to different environments and resistance to barrenness make it an excellent forage and ecological restoration plant. However, its molecular genetics and genomic relationships among populations are yet to be uncovered. RESULT: Here we report on a genomic variation map from worldwide 272 L. corniculatus accessions by genome resequencing. Our analysis suggests that L. corniculatus accessions have high genetic diversity and could be further divided into three subgroups, with the genetic diversity centers were located in Transcaucasia. Several candidate genes and SNP site associated with CNglcs content and growth traits were identified by genome-wide associated study (GWAS). A non-synonymous in LjMTR was responsible for the decreased expression of CNglcs synthesis genes and LjZCD was verified to positively regulate CNglcs synthesis gene CYP79D3. The LjZCB and an SNP in LjZCA promoter were confirmed to be involved in plant growth. CONCLUSION: This study provided a large number of genomic resources and described genetic relationship and population structure among different accessions. Moreover, we attempt to provide insights into the molecular studies and breeding of CNglcs and growth traits in L. corniculatus.

MeJA-responsive bHLH transcription factor LjbHLH7 regulates cyanogenic glucoside biosynthesis in Lotus japonicus.

Cyanogenic glucosides (CNglcs) play an important role in plant defense response; however, the mechanism of regulation of CNglc synthesis by the external environment and endogenous hormones is largely unclear. In this study, we found that jasmonates (JAs) promoted the synthesis of CNglcs by activating the expression of CNglc biosynthesis genes in Lotus japonicus. Several differentially expressed basic helix-loop-helix (bHLH) family genes related to the synthesis of CNglcs were identified by RNA-seq. LjbHLH7 can directly activate the expression of CYP79D3 gene, the first step of CNglc synthesis, by binding to the G-box sequence of its promoter. Transgenic plants overexpressing LjbHLH7 exhibited higher relative CNglc content and enhanced insect resistance compared with the wild type. Furthermore, the transcriptional activity of LjbHLH7 was suppressed by the interaction with the L. japonicus JASMONATE-ZIM DOMAIN protein LjJAZ4. Based on these results, we propose that LjbHLH7 acts as an activator and LjJAZ4 acts as a repressor of JA-induced regulation of CNglc biosynthesis in L. japonicus.

Agrobacterium-Mediated Transformation of Yeast and Fungi.

Two decades ago, it was discovered that the well-known plant vector Agrobacterium tumefaciens can also transform yeasts and fungi when these microorganisms are co-cultivated on a solid substrate in the presence of a phenolic inducer such as acetosyringone. It is important that the medium has a low pH (5-6) and that the temperature is kept at room temperature (20-25 °C) during co-cultivation. Nowadays, Agrobacterium-mediated transformation (AMT) is the method of choice for the transformation of many fungal species; as the method is simple, the transformation efficiencies are much higher than with other methods, and AMT leads to single-copy integration much more frequently than do other methods. Integration of T-DNA in fungi occurs by non-homologous end-joining (NHEJ), but also targeted integration of the T-DNA by homologous recombination (HR) is possible. In contrast to AMT of plants, which relies on the assistance of a number of translocated virulence (effector) proteins, none of these (VirE2, VirE3, VirD5, VirF) are necessary for AMT of yeast or fungi. This is in line with the idea that some of these proteins help to overcome plant defense. Importantly, it also showed that VirE2 is not necessary for the transport of the T-strand into the nucleus. The yeast Saccharomyces cerevisiae is a fast-growing organism with a relatively simple genome with reduced genetic redundancy. This yeast species has therefore been used to unravel basic molecular processes in eukaryotic cells as well as to elucidate the function of virulence factors of pathogenic microorganisms acting in plants or animals. Translocation of Agrobacterium virulence proteins into yeast was recently visualized in real time by confocal microscopy. In addition, the yeast 2-hybrid system, one of many tools that have been developed for use in this yeast, was used to identify plant and yeast proteins interacting with the translocated Agrobacterium virulence proteins. Dedicated mutant libraries, containing for each gene a mutant with a precise deletion, have been used to unravel the mode of action of some of the Agrobacterium virulence proteins. Yeast deletion mutant collections were also helpful in identifying host factors promoting or inhibiting AMT, including factors involved in T-DNA integration. Thus, the homologous recombination (HR) factor Rad52 was found to be essential for targeted integration of T-DNA by HR in yeast. Proteins mediating double-strand break (DSB) repair by end-joining (Ku70, Ku80, Lig4) turned out to be essential for non-homologous integration. Inactivation of any one of the genes encoding these end-joining factors in other yeasts and fungi was employed to reduce or totally eliminate non-homologous integration and promote efficient targeted integration at the homologous locus by HR. In plants, however, their inactivation did not prevent non-homologous integration, indicating that T-DNA is captured by different DNA repair pathways in plants and fungi.

pXST, a novel vector for TA cloning and blunt-end cloning.

BACKGROUND: With the rapid development of sequencing technologies, increasing amount of genomic information has been accumulated. To clone genes for further functional studies in large scale, a cheap, fast and efficient cloning vector is desired. RESULTS: A bifunctional vector pXST has been constructed. The pXST vector harbors a XcmI-ccdB-XcmI cassette and restriction site SmaI. Digestion the vector with XcmI generates a single thymidine (T) overhang at 3' end which facilitates TA cloning, and SmaI gives blunt end that enables the blunt-end ligation. Multiple products with various sizes were amplified from cassava genome by PCR and each PCR fragment was separately cloned into a pXST using TA cloning and blunt-end ligation methods. In general, the TA cloning gave higher transformation efficiency than blunt-end ligation for inserts with all different sizes, and the transformation efficiency significantly decreased with increasing size of inserts. The highest transformation efficiency (8.6 × 106 transformants/µg) was achieved when cloning 517 bp DNA fragment using TA cloning. No significant difference observed in the positive cloning efficiency between two ligation methods and the positive cloning efficiency could reach as high as 100% especially for small inserts (e.g. 517 and 957 base pairs). CONCLUSIONS: We describe a simple and general method to construct a novel pXST vector. We confirm the feasibility of using pXST vector to clone PCR products amplified from cassava genome with both TA cloning and blunt-end ligation methods. The pXST plasmid has several advantages over many currently available vectors in that (1) it possesses XcmI-ccdB-XcmI cassette and restriction site SmaI, enabling both TA cloning and blunt-end ligation. (2) it allows direct selection of positive recombinant plasmids in Escherichia coli through disruption of the ccdB gene. (3) it improves positive cloning efficiency by introducing the ccdB gene, reducing the possibility of self-ligation from insufficient digested plasmids. (4) it could be used by high performance and cost-effective cloning methods. Therefore, this dual function vector would offer flexible alternatives for gene cloning experiments to researchers.

The Agrobacterium tumefaciens virulence protein VirE3 is a transcriptional activator of the F-box gene VBF.

During Agrobacterium tumefaciens-mediated transformation of plant cells a part of the tumour-inducing plasmid, T-DNA, is integrated into the host genome. In addition, a number of virulence proteins are translocated into the host cell. The virulence protein VirE3 binds to the Arabidopsis thaliana pBrp protein, a plant-specific general transcription factor of the TFIIB family. To study a possible role for VirE3 in transcriptional regulation, we stably expressed virE3 in A. thaliana under control of a tamoxifen-inducible promoter. By RNA sequencing we showed that upon expression of virE3 the RNA levels of 607 genes were increased more than three-fold and those of 132 genes decreased more than three-fold. One of the strongly activated genes was that encoding VBF (At1G56250), an F-box protein that may affect the levels of the VirE2 and VIP1 proteins. Using Arabidopsis cell suspension protoplasts we showed that VirE3 stimulates the VBF promoter, especially when co-expressed with pBrp. Although pBrp is localized at the external surface of plastids, co-expression of VirE3 and pBrp in Arabidopsis cell suspension protoplasts resulted in the accumulation of pBrp in the nucleus. Our results suggest that VirE3 affects the transcriptional machinery of the host cell to favour the transformation process.

Systematic analysis of the roles of c-di-GMP signaling in Xanthomonas oryzae pv. oryzae virulence.

Cyclic di-guanosine monophosphate (c-di-GMP) is a ubiquitous second messenger, i.e. essential to bacterial adaptation to environments. Cellular c-di-GMP level is regulated by the diguanylate cyclases and the phosphodiesterases, and the signal transduction depends on its receptors. In Xanthomonas oryzae pv. oryzae strain PXO99A, 37 genes were predicted to encode GGDEF, EAL, GGDEF/EAL, HD-GYP, FleQ, MshE, PilZ, CuxR, Clp, and YajQ proteins that may be involved in c-di-GMP turnover or function as c-di-GMP receptors. Although the functions of some of these genes have been studied, but the rest have not been extensively studied. Here, we deleted these 37 genes from PXO99A and analyzed the virulence, motility, biofilm, and EPS production of these mutants. Our results show that most of these genes are required for PXO99A virulence, motility, biofilm formation, or exopolysaccharide production. Although some of them have been reported in previous studies, we found four novel genes (gedpX8, gdpX11, pliZX4, and yajQ) are implicated in X. oryzae pv. oryzae virulence. Our data demonstrate that c-di-GMP signaling is vital for X. oryzae pv. oryzae virulence and some virulence-related factors production, but there is no positive correlation between them in most cases. Taken together, our systematic research provides a new light to understand the c-di-GMP signaling network in X. oryzae pv. oryzae.

Transcriptomic profiling suggests candidate molecular responses to waterlogging in cassava.

Owing to climate change impacts, waterlogging is a serious abiotic stress that affects crops, resulting in stunted growth and loss of productivity. Cassava (Manihot esculenta Grantz) is usually grown in areas that experience high amounts of rainfall; however, little research has been done on the waterlogging tolerance mechanism of this species. Therefore, we investigated the physiological responses of cassava plants to waterlogging stress and analyzed global gene transcription responses in the leaves and roots of waterlogged cassava plants. The results showed that waterlogging stress significantly decreased the leaf chlorophyll content, caused premature senescence, and increased the activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) in the leaves and roots. In total, 2538 differentially expressed genes (DEGs) were detected in the leaves and 13364 in the roots, with 1523 genes shared between the two tissues. Comparative analysis revealed that the DEGs were related mainly to photosynthesis, amino metabolism, RNA transport and degradation. We also summarized the functions of the pathways that respond to waterlogging and are involved in photosynthesis, glycolysis and galactose metabolism. Additionally, many transcription factors (TFs), such as MYBs, AP2/ERFs, WRKYs and NACs, were identified, suggesting that they potentially function in the waterlogging response in cassava. The expression of 12 randomly selected genes evaluated via both quantitative real-time PCR (qRT-PCR) and RNA sequencing (RNA-seq) was highly correlated (R2 = 0.9077), validating the reliability of the RNA-seq results. The potential waterlogging stress-related transcripts identified in this study are representatives of candidate genes and molecular resources for further understanding the molecular mechanisms underlying the waterlogging response in cassava.

Genome-wide identification and expression of TIFY family in cassava (Manihot esculenta Crantz).

Plant-specific TIFY [TIF(F/Y)XG] proteins serve important roles in the regulation of plant stress responses. This family encodes four subfamilies of proteins, JAZ (JASMONATE ZIM-domain), PPD (PEAPOD), ZML (Zinc-finger Inflorescence-like), and TIFY. In this work, a total of 16 JAZ, 3 PPD, 7 ZML, and 2 TIFY genes were found in cassava (Manihot esculenta Crantz) at the genome-wide level. The phylogenetics, exon-intron structure, motif organization, and conserved domains of these genes were analyzed to characterize the members of the JAZ, PPD, and ZML subfamilies. Chromosome location and synteny analyses revealed that 26 JAZ, PPD, and ZML genes were irregularly distributed across 14 of the 18 chromosomes, and 18 gene pairs were implicated in large-scale interchromosomal segmental duplication events. In addition, JAZ, PPD, and ZML gene synteny comparisons between cassava and three other plant species (Arabidopsis, Populus trichocarpa, and rice) uncovered vital information about their likely evolution. The prediction of protein interaction network and cis-acting elements reveal the function of JAZ, PPD, and ZML genes. Subsequently, expression patterns of JAZ, PPD, and ZML genes were validated by qRT-PCR as being expressed in response to osmotic, salt, and cadmium stress. Moreover, almost all JAZ subfamily genes were responsive to jasmonic acid (JA) treatment. In particular, MeJAZ1, MeJAZ13, and MeJAZ14, were highly up-regulated by three treatments, and these genes may deserve further study. This comprehensive study lays the groundwork for future research into TIFY family genes in cassava and may be valuable for genetic improvement of cassava and other related species.

The RavA/VemR two-component system plays vital regulatory roles in the motility and virulence of Xanthomonas campestris.

Xanthomonas campestris pv. campestris (Xcc) can cause black rot in cruciferous plants worldwide. Two-component systems (TCSs) are key for bacterial adaptation to various environments, including hosts. VemR is a TCS response regulator and crucial for Xcc motility and virulence. Here, we report that RavA is the cognate histidine kinase (HK) of VemR and elucidate the signalling pathway by which VemR regulates Xcc motility and virulence. Genetic analysis showed that VemR is epistatic to RavA. Using bacterial two-hybrid experiments and pull-down and phosphorylation assays, we found that RavA can interact with and phosphorylate VemR, suggesting that RavA is the cognate HK of VemR. In addition, we found that RpoN2 and FleQ are epistatic to VemR in regulating bacterial motility and virulence. In vivo and in vitro experiments demonstrated that VemR interacts with FleQ but not with RpoN2. RavA/VemR regulates the expression of the flagellin-encoding gene fliC by activating the transcription of the rpoN2-vemR-fleQ and flhF-fleN-fliA operons. In summary, our data show that the RavA/VemR TCS regulates FleQ activity and thus influences the expression of motility-related genes, thereby affecting Xcc motility and virulence. The identification of this novel signalling pathway will deepen our understanding of Xcc-plant interactions.

JAZ8 Interacts With VirE3 Attenuating Agrobacterium Mediated Root Tumorigenesis.

Agrobacterium tumefaciens can cause crown gall tumors by transferring both an oncogenic piece of DNA (T-DNA) and several effector proteins into a wide range of host plants. For the translocated effector VirE3 multiple functions have been reported. It acts as a transcription factor in the nucleus binding to the Arabidopsis thaliana pBrp TFIIB-like protein to activate the expression of VBF, an F-box protein involved in degradation of the VirE2 and VIP1 proteins, facilitating Agrobacterium-mediated transformation. Also VirE3 has been found at the plasma membrane, where it could interact with VirE2. Here, we identified AtJAZ8 in a yeast two-hybrid screening with VirE3 as a bait and confirmed the interaction by pull-down and bimolecular fluorescence complementation assays. We also found that the deletion of virE3 reduced Agrobacterium virulence in a root tumor assay. Overexpression of virE3 in Arabidopsis enhanced tumorigenesis, whereas overexpression of AtJAZ8 in Arabidopsis significantly decreased the numbers of tumors formed. Further experiments demonstrated that AtJAZ8 inhibited the activity of VirE3 as a plant transcriptional regulator, and overexpression of AtJAZ8 in Arabidopsis activated AtPR1 gene expression while it repressed the expression of AtPDF1.2. Conversely, overexpression of virE3 in Arabidopsis suppressed the expression of AtPR1 whereas activated the expression of AtPDF1.2. Our results proposed a novel mechanism of counter defense signaling pathways used by Agrobacterium, suggesting that VirE3 and JAZ8 may antagonistically modulate the salicylic acid/jasmonic acid (SA/JA)-mediated plant defense signaling response during Agrobacterium infection.

Diversity of the Bacterial Microbiome Associated With the Endosphere and Rhizosphere of Different Cassava (Manihot esculenta Crantz) Genotypes.

Root-associated microbial communities play important roles in plant growth and development. However, little attention has been paid to the microbial community structures associated with cassava, which is a staple food for approximately 800 million people worldwide. Here, we studied the diversity and structure of tuber endosphere and rhizosphere bacterial communities in fourteen cassava genotypes: SC5, SC8, SC9, SC205, KU50, R72, XL1, FX01, SC16, 4612, 587, 045, S0061, and 1110. The results of bacterial 16S rDNA sequencing showed that the richness and diversity of bacteria in the rhizosphere were higher than those in the tuber endosphere across the 14 cassava genotypes. After sequencing, 21 phyla and 310 genera were identified in the tuberous roots, and 36 phyla and 906 genera were identified in the rhizosphere soils. The dominant phylum across all tuber samples was Firmicutes, and the dominant phyla across all rhizosphere samples were Actinobacteria, Proteobacteria, and Acidobacteria. The numbers of core bacterial taxa within the tuber endospheres and the rhizospheres of all cassava genotypes were 11 and 236, respectively. Principal coordinate analysis and hierarchical cluster analysis demonstrated significant differences in the compositions of rhizosphere soil microbiota associated with the different cassava genotypes. Furthermore, we investigated the metabolic changes in tuber roots of three genotypes, KU50, SC205, and SC9. The result showed that the abundances of Firmicutes, Proteobacteria, and Actinobacteria in tuber samples were positively correlated with organic acids and lipids and negatively correlated with vitamins and cofactors. These results strongly indicate that there are clear differences in the structure and diversity of the bacterial communities associated with different cassava genotypes.

A denoising and enhancing method framework for 4D ultrasound images of human fetal heart.

BACKGROUND: 4D ultrasound images of human fetal heart are important for medical applications such as evaluation of fetal heart function and early diagnosis of congenital heart diseases. However, due to the high noise and low contrast characteristics in fetal ultrasound images, denoising and enhancements are important. METHODS: In this paper, a special method framework for denoising and enhancing is proposed. It consists of a 4D-NLM (non-local means) denoising method for 4D fetal heart ultrasound image sequence, which takes advantage of context similar information in neighboring images to denoise the target image, and an enhancing method called the Adaptive Clipping for Each Histogram Pillar (ACEHP), which is designed to enhance myocardial spaces to distinguish them from blood spaces. RESULTS: Denoising and enhancing experiments show that 4D-NLM method has better denoising effect than several classical and state-of-the-art methods such as NLM and WNNM. Similarly, ACEHP method can keep noise level low while enhancing myocardial regions better than several classical and state-of-the-art methods such as CLAHE and SVDDWT. Furthermore, in the volume rendering after the combined "4D-NLM+ACEHP" processing, the cardiac lumen is clear and the boundary is neat. The Entropy value that can be achieved by our method framework (4D-NLM+ACEHP) is 4.84. CONCLUSIONS: Our new framework can thus provide important improvements to clinical fetal heart ultrasound images.

A Novel Glycerol Kinase Gene OsNHO1 Regulates Resistance to Bacterial Blight and Blast Diseases in Rice.

Glycerol-induced resistance to various pathogens has been reported in different plants. Glycerol kinase (GK), a vital rate-limiting enzyme that catalyzes glycerol conversion to glycerol-3-phosphate (G3P), participates in responses to both abiotic and biotic stresses. However, its physiological importance in rice defenses against pathogens remains unclear. In this research, quantification analysis revealed that GK levels were significantly induced in rice leaves infected by Xanthomonas oryzae pv. oryzae (Xoo) strain PXO99. A typical GK-encoding gene OsNHO1 was cloned in rice. The transcriptional levels of OsNHO1 were significantly induced by salicylic acid, jasmonic acid, and Xoo-PXO99. Ectopic expression of OsNHO1 partially rescued the resistance to P. s. pv. phaseolicola in the Arabidopsis nho1 mutant. In the overexpressing transgenic rice lines (OsNHO1-OE), the content of GK and the transcriptional level of OsNHO1 were increased and the resistance to bacterial blight and blast was improved, while reduced OsNHO1 expression impaired the resistance in OsNHO1-RNAi lines. The wax contents and expression of the wax synthesis regulatory genes were significantly increased in the overexpression lines but decreased in the OsNHO1-RNAi lines. We then confirmed the interaction partner of OsNHO1 using yeast two-hybrid and bimolecular fluorescence complementation assays. The transcription of the interaction partner-encoding genes OsSRC2 and OsPRs in OsNHO1-RNAi lines was downregulated but upregulated in OsNHO1-OE lines. Thus, we concluded that OsNHO1 provided disease resistance by affecting the wax content and modulating the transcription levels of PR genes.

Direct 3D model extraction method for color volume images.

BACKGROUND: There is a great demand for the extraction of organ models from three-dimensional (3D) medical images in clinical medicine diagnosis and treatment. OBJECTIVE: We aimed to aid doctors in seeing the real shape of human organs more clearly and vividly. METHODS: The method uses the minimum eigenvectors of Laplacian matrix to automatically calculate a group of basic matting components that can properly define the volume image. These matting components can then be used to build foreground images with the help of a few user marks. RESULTS: We propose a direct 3D model segmentation method for volume images. This is a process of extracting foreground objects from volume images and estimating the opacity of the voxels covered by the objects. CONCLUSIONS: The results of segmentation experiments on different parts of human body prove the applicability of this method.

3D Shared Matting Method for Directly Extracting Standard Organ Models from Human Body Color Volume Image.

BACKGROUND: In some medical applications (e.g., virtual surgery), standard human organ models are very important and useful. Now that real human body slice image sets have been collected by several countries, it is possible to obtain real standard organ models. INTRODUCTION: Understanding how to abandon the traditional model construction method of Photoshop sketching slice by slice and directly extracting 3D models from volume images has been an interesting and challenging issue. In this paper, a 3D color volume image matting method has been proposed to segment human body organ models. METHODS: First, the scope of the known area will be expanded by means of propagation. Next, neighborhood sampling to find the best sampling for voxels in an unknown region will be performed and then the preliminary opacity using the sampling results will be calculated. RESULTS: The final result will be obtained by applying local smoothing to the image. CONCLUSION: From the experimental results, it has been observed that our method is effective for real standard organ model extraction.

RaxM regulates the AvrXa21 (RaxX)-mediated immune response.

Xanthomonas oryzae pv. oryzae (Xoo) is the causal agent of rice bacterial blight disease, which causes a reduction in rice production. The interaction between Xoo and rice is a model for the study of the gene-for-gene hypothesis, in which a resistance (R) gene encoding a product interacts with an effector molecule encoded by a corresponding bacterial avirulence (avr) gene. Rice XA21 functions as a plant innate immune receptor (R protein) and recognizes the avirulence protein (RaxX) of Xoo to induce the immune response and cope with pathogen attack. The sulphuration of RaxX by the tyrosine sulphotransferase RaxST is essential to its activity. The expression of raxST is regulated by the RaxH/RaxR and phoP/phoQ two-component systems. However, the regulation of raxX expression remains unclear. Here, we showed that a gene (raxM) encodes a small protein, which functions as a regulator of raxX expression. raxX and raxM are located upstream of raxST. Transcriptional analysis indicates that raxX and raxM are separately transcribed and the promoter of raxX is located at the raxM coding region. The RaxM protein regulates its own and raxX expression, and is required for the XA21-mediated immunity response. Therefore, we identified a regulator of raxX expression and of the Xoo-rice interaction. Our findings suggest that RaxX is not only regulated at the post-translational level, but also at the transcriptional level.

MeBIK1, a novel cassava receptor-like cytoplasmic kinase, regulates PTI response of transgenic Arabidopsis.

Cassava bacterial blight is the most destructive disease in cassava, causing a significant reduction in its production. The innate immunity response, which has a broad spectrum and a persistent effect, is the basal defence of plants in response to pathogens. Isolation and identification of innate immune-related genes in cassava will contribute to understanding the disease resistance mechanism. In Arabidopsis, the receptor-like cytoplasmic kinase (RLCK) AtBIK1 is known to be an important signal mediator in pathogen-associated molecular pattern-triggered immunity (PTI) response, forming a signal complex from various receptors including the flagellin receptor FLS2, the chitin receptor CERK1 and the receptor for bacterial EF-Tu EFR (Zhang et al. 2010). In the present study, we selected a candidate receptor-like cytoplasmic kinase gene, MeBIK1, from the cassava genome. MeBIK1 encodes a 409 amino acid polypeptide comprising a typical serine/threonine protein kinase domain, and is located on the cell membrane. MeBIK1 gene expression was significantly increased upon stimulation with flagellin (flg22) and peaked at 1h. In vitro genetic complementation experiment showed that MeBIK1 complemented the reduced pathogen-associated molecular pattern-triggered immunity (PTI) response in Arabidopsis bik1 mutant. Arabidopsis MeBIK1 overexpression lines OX1 demonstrated a strong resistance to Xanthomonas axonopodis pv. manihotis HN01, whereas its sensitivity to Pseudomonas syringae pv. tomato DC3000 was enhanced. The peak level of reactive oxygen species (ROS) burst was reached in different Arabidopsis plants (bik1, OX1 and wild type) at 12min after induction with flg22. However, the OX1 showed significantly higher ROS levels than the control and mutant, whereas the lowest level of ROS burst was found in the bik1 mutant. These results indicate that cassava MeBIK1 has a similar function as Arabidopsis AtBIK1 and improves disease resistance in transgenic Arabidopsis by regulating the PTI response.