OsMPK12 positively regulates rice blast resistance via OsEDC4-mediated transcriptional regulation of immune-related genes.

Mitogen-activated protein kinase (MAPK) signalling cascades are functionally important signalling modules in eukaryotes. Transcriptome reprogramming of immune-related genes is a key process in plant immunity. Emerging evidence shows that plant MAPK cascade is associated with processing (P)-body components and contributes to transcriptome reprogramming of immune-related genes. However, it remains largely unknown how this process is regulated. Here, we show that OsMPK12, which is induced by Magnaporthe oryzae infection, positively regulates rice blast resistance. Further analysis revealed that OsMPK12 directly interacts with enhancer of mRNA decapping protein 4 (OsEDC4), a P-body-located protein, and recruits OsEDC4 to where OsMPK12 is enriched. Importantly, OsEDC4 directly interacts with two decapping complex members OsDCP1 and OsDCP2, indicating that OsEDC4 is a subunit of the mRNA decapping complex. Additionally, we found that OsEDC4 positively regulates rice blast resistance by regulating expression of immune-related genes and maintaining proper mRNA levels of some negatively-regulated genes. And OsMPK12 and OsEDC4 are also involved in rice growth and development regulation. Taken together, our data demonstrate that OsMPK12 positively regulates rice blast resistance via OsEDC4-mediated mRNA decay of immune-related genes, providing new insight into not only the new role of the MAPK signalling cascade, but also posttranscriptional regulation of immune-related genes.

Receptor-like kinases OsRLK902-1 and OsRLK902-2 form immune complexes with OsRLCK185 to regulate rice blast resistance.

Receptor-like kinases (RLKs) are major regulators of the plant immune response and play important roles in the perception and transmission of immune signals. RECEPTOR LIKE KINASE 902 (RLK902) is at the key node in leucine-rich repeat receptor-like kinase interaction networks and positively regulates resistance to the bacterial pathogen Pseudomonas syringae in Arabidopsis. However, the function of RLK902 in fungal disease resistance remains obscure. In this study, we found that the expression levels of OsRLK902-1 and OsRLK902-2, encoding two orthologues of RLK902 in rice, were induced by Magnaporthe oryzae, chitin, and flg22 treatment. osrlk902-1 and osrlk902-2 knockout mutants displayed enhanced susceptibility to M. oryzae. Interestingly, the osrlk902-1 rlk902-2 double mutant exhibited similar disease susceptibility, hydrogen peroxide production, and callose deposition to the two single mutants. Further investigation showed that OsRLK902-1 interacts with and stabilizes OsRLK902-2. The two OsRLKs form a complex with OsRLCK185, a key regulator in chitin-triggered immunity, and stabilize it. Taken together, our data demonstrate that OsRLK902-1 and OsRLK902-2, as well as OsRLCK185 function together in regulating disease resistance to M. oryzae in rice.

The 14-3-3 protein GF14c positively regulates immunity by modulating the protein homoeostasis of the GRAS protein OsSCL7 in rice.

Various types of transcription factors have been reported to be involved in plant-pathogen interactions by regulating defence-related genes. GRAS proteins, plant- specific transcription factors, have been shown to play essential roles in plant growth, development and stress responses. By performing a transcriptome study on rice early defence responses to Magnaporthe oryzae, we identified a GRAS protein, OsSCL7, which was induced by M. oryzae infection. We characterized the function of OsSCL7 in rice disease resistance. OsSCL7 was upregulated upon exposure to M. oryzae and pathogen-associated molecular pattern treatments, and knocking out OsSCL7 resulted in decreased disease resistance of rice to M. oryzae. In contrast, overexpression of OsSCL7 could improve rice disease resistance to M. oryzae. OsSCL7 was mainly localized in the nucleus and showed transcriptional activity. OsSCL7 can interact with GF14c, a 14-3-3 protein, and loss-of-function GF14c leads to enhanced susceptibility to M. oryzae. Additionally, OsSCL7 protein levels were reduced in the gf14c mutant and knocking out OsSCL7 affected the expression of a series of defence-related genes. Taken together, these findings uncover the important roles of OsSCL7 and GF14c in plant immunity and a potential mechanism by which plants fine-tune immunity by regulating the protein stability of a GRAS protein via a 14-3-3 protein.

OsExo70B1 Positively Regulates Disease Resistance to Magnaporthe oryzae in Rice.

The exocyst, an evolutionarily conserved octameric protein complex, mediates tethering of vesicles to the plasma membrane in the early stage of exocytosis. Arabidopsis Exo70, a subunit of the exocyst complex, has been found to be involved in plant immunity. Here, we characterize the function of OsExo70B1 in rice. OsExo70B1 mainly expresses in leaf and shoot and its expression is induced by pathogen-associated molecular patterns (PAMPs) and rice blast fungus Magnaporthe oryzae (M. oryzae). Knocking out OsExo70B1 results in significantly decreased resistance and defense responses to M. oryzae compared to the wild type, including more disease lesions and enhanced fungal growth, downregulated expression of pathogenesis-related (PR) genes, and decreased reactive oxygen species accumulation. In contrast, the exo70B1 mutant does not show any defects in growth and development. Furthermore, OsExo70B1 can interact with the receptor-like kinase OsCERK1, an essential component for chitin reception in rice. Taken together, our data demonstrate that OsExo70B1 functions as an important regulator in rice immunity.

Dwarf and deformed flower 1, encoding an F-box protein, is critical for vegetative and floral development in rice (Oryza sativa L.).

Recent studies have shown that F-box proteins constitute a large family in eukaryotes, and play pivotal roles in regulating various developmental processes in plants. However, their functions in monocots are still obscure. In this study, we characterized a recessive mutant dwarf and deformed flower 1-1 (ddf1-1) in Oryza sativa (rice). The mutant is abnormal in both vegetative and reproductive development, with significant size reduction in all organs except the spikelet. DDF1 controls organ size by regulating both cell division and cell expansion. In the ddf1-1 spikelet, the specification of floral organs in whorls 2 and 3 is altered, with most lodicules and stamens being transformed into glume-like organs and pistil-like organs, respectively, but the specification of lemma/palea and pistil in whorls 1 and 4 is not affected. DDF1 encodes an F-box protein anchored in the nucleolus, and is expressed in almost all vegetative and reproductive tissues. Consistent with the mutant floral phenotype, DDF1 positively regulates B-class genes OsMADS4 and OsMADS16, and negatively regulates pistil specification gene DL. In addition, DDF1 also negatively regulates the Arabidopsis LFY ortholog APO2, implying a functional connection between DDF1 and APO2. Collectively, these results revealed that DDF1, as a newly identified F-box gene, is a crucial genetic factor with pleiotropic functions for both vegetative growth and floral organ specification in rice. These findings provide additional insights into the molecular mechanism controlling monocot vegetative and reproductive development.

TRAIL suppresses tumor growth in mice by inducing tumor-infiltrating CD4(+)CD25 (+) Treg apoptosis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a promising and novel anticancer cytokine, specifically kills numerous tumor cells by apoptosis. However, some malignancies are resistant to TRAIL treatment in clinical trials, thus limiting its therapeutic potential. In the present study, the TRAIL-resistant murine hepatocellular carcinoma cell line Hepa1-6 was used to elucidate the physiological significance of TRAIL resistance, especially with respect to the immune regulatory function of TRAIL. Hepa1-6 cells were resistant to TRAIL-induced apoptosis in vitro; however, intratumoral injection of recombinant soluble TRAIL inhibited tumor growth and prolonged survival time in tumor-bearing mice. Local TRAIL treatment decreased the number of intratumoral CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) but did not affect CD4(+)CD25(+)Foxp3(+) Tregs in the draining lymph nodes and spleen. Further investigation showed that TRAIL induced apoptosis of tumor-activated CD4(+)CD25(+)Foxp3(+) Tregs, but not of CD4(+)CD25(-) T cells. Moreover, mouse TRAIL receptor DR5 expression was detected on the surface of the tumor-infiltrating CD4(+)CD25(+)Foxp3(+) Tregs, but not on naïve CD4(+)CD25(+)Foxp3(+) Tregs. Interestingly, intratumoral injection of TRAIL not only decreased the number of CD4(+)CD25(+)Foxp3(+) Tregs but also increased the number of tumor-specific CD8(+) CTL and augmented their cytotoxicity to the tumor cells. These data provide the novel evidence for an immune regulatory function of TRAIL and may shed light on the clinical application of TRAIL.

Epirubicin potentiates recombinant adeno-associated virus type 2/5-mediated TRAIL expression in fibroblast-like synoviocytes and augments the antiarthritic effects of rAAV2/5-TRAIL.

OBJECTIVE: Synovial cells in rheumatoid synovium display abnormal proliferation, which leads to joint destruction. TRAIL has been described as a proapoptotic factor in fibroblast-like synoviocytes (FLS). This study was undertaken to investigate the functions of rAAV2/5-TRAIL in human FLS and in arthritic mice. METHODS: Primary human FLS were infected with rAAV2/5-TRAIL in the presence or absence of epirubicin. Transgene expression was monitored by both enzyme-linked immunosorbent assay and flow cytometry. The disease-modulating activity of epirubicin plus rAAV2/5-TRAIL was investigated in mice with collagen-induced arthritis (CIA). RESULTS: Subtoxic doses of epirubicin potentiated rAAV2/5-mediated TRAIL expression in FLS and simultaneously enhanced the sensitivity of FLS to TRAIL. Epirubicin treatment up-regulated death receptor 4 (DR-4) and DR-5 expression and down-regulated FLIP expression, thereby enhancing the activation of procaspase 3, procaspase 8, and procaspase 9. An in vivo study showed that the combination of rAAV2/5-TRAIL gene therapy and epirubicin chemotherapy provided augmented antiarthritic effects in a mouse model of CIA. The intraarticular injection of rAAV2/5-TRAIL combined with epirubicin treatment significantly reduced the severity and incidence of CIA and joint swelling in the animals. Histologic evaluations revealed that inflammatory cell infiltration, cartilage destruction, and bone erosion were significantly reduced in the joints of the mice receiving the synthetic treatment. Results of a viral genome copy number assay indicated that epirubicin dramatically augmented the expression of rAAV2/5-TRAIL without altering its tissue distribution. CONCLUSION: These results suggest that epirubicin enhances the antiarthritic effect of rAAV2/5-TRAIL and that combination treatment might be an important therapeutic alternative, with practical significance for rheumatoid arthritis.

Characterization of Osmads6-5, a null allele, reveals that OsMADS6 is a critical regulator for early flower development in rice (Oryza sativa L.).

AGL6-clade genes are a subfamily of MADS-box genes and preferentially expressed in floral organs. OsMADS6 and OsMADS17 are two AGL6-like genes in rice. OsMADS17 has been shown to play a minor role in floral development and appears to result from a duplication of OsMADS6. OsMADS6 was initially named as MFO1 for mosaic floral organs based on its moderate mutant phenotypes. So far, four moderate or weak mutant alleles of OsMADS6 have been described, providing valuable insights into its role in flower development. Here, we report a null allele of OsMADS6 (Osmads6-5), which exhibited a strong mutant phenotype in spikelet without affecting vegetative traits, causing all floral organs except lemma homeotically transformed into lemma-like organs (LLOs) as well as an indeterminate floral meristem, thus resulting in a mutant floret consisting of reiterating whorls of lemma and LLOs. In consistently, over-expression of OsMADS6 led to additional lodicule-, stamen- and carpel-like organs. Expression analysis showed that OsMADS6 controls the formation of the incipient primordia of lodicule, stamen and carpel via regulating the expression of class B, C and SEP-like MADS-box genes. Taken together, our results revealed that OsMADS6 acts as a critical regulator for early flower development in rice and provide novel insights into the molecular mechanism of OsMADS6.

Molecular cloning and functional characterization of OsJAG gene based on a complete-deletion mutant in rice (Oryza sativa L.).

In this article, we report an independent work of positional cloning and functional characterization of OsJAG gene in rice. The merit of our work is that we used a genuine null mutant, in which the wild-type allele was completely deleted. This allowed us to identify the mutant phenotypes accurately without the interference of residual function of the target gene. OsJAG is an important gene with pleiotropy, expressing almost throughout the plant and acting in both vegetative phase and reproductive phase. But its main and crucial roles are in regulating the development of all floral organs, especially in specifying the identity of stamens. Interestingly, OsJAG does not affect the number of floral organ primordial and so of floral organs in each whorl, suggesting that OsJAG does not influence the initiation of floral organ primordia, but affect the developmental fate of all floral organs after their primordia have initiated. Loss of OsJAG function results in maldevelopment of all floral organs, such as degenerated lemma and palea, elongated lodicules and deformed and sterile pistil. The stamen appears to be more sensitive to the mutation. All the six stamens in a mutant floret were thoroughly transformed into six pistil-like organs developed at the presumptive positions of the stamens in whorl 3.

Evaluation of specific delivery of chimeric phi29 pRNA/siRNA nanoparticles to multiple tumor cells.

The pRNA (packaging RNA) of bacteriophage phi29 DNA packaging motor has been reported to have novel applications in nanotechnology and nanomedicine. The unique ability of pRNA to form dimers, trimers, hexamers and patterned superstructures via the interaction of two reengineered interlocking loops makes it a promising polyvalent vehicle to load siRNA and other therapeutic molecules and be applied as a therapeutic nanoparticle in tumor therapy. In this study, several tumor cell lines were used to evaluate the previously reported pRNA nanotechnology for specific siRNA delivery and for the silencing of targeted genes. It was found that MCF-7 and HeLa cells, out of twenty-five tested tumor cell lines, expressed high levels of folate receptors and exhibited specific binding of the FITC-folate-pRNA nanoparticles, while the others expressed low levels and thus, for these, delivery was not feasible using folate as a targeting agent. Folate receptor positive tumor cells were then incubated with the chimeric pRNA dimer harboring both the folate-pRNA and the chimeric pRNA/siRNA (survivin). Knock down effects of survivin expression in these tumor cells were detected at the mRNA level by real time-PCR and at the protein level by western blot. Apoptosis was detected by flow cytometry analysis with dual staining of annexinV-FITC and PI. The data suggest that the chimeric pRNA nanoparticles containing folate-pRNA and pRNA/siRNA (survivin) could be specifically taken up by tumor cells through folate receptor-mediated endocytosis, resulting in significant inhibition of both transcription and expression of survivin in tumor cells and triggering cell apoptosis. Using such protein-free nanoparticles as therapeutic reagents would not only allow specific gene delivery and extend the in vivo retaining time but also allow long-term administration of therapeutic particles, therefore avoiding the induction of antibodies caused by repeated treatment for chronic diseases.

Fabrication of Polyvalent Therapeutic RNA Nanoparticles for Specific Delivery of siRNA, Ribozyme and Drugs to Targeted Cells for Cancer Therapy.

Bacteriophage phi29 DNA packaging motor is geared by a six-pRNA ring. pRNA is able to form a multimeric complex and patterned superstructures via the interaction of two reengineered interlocking loops. This unique feature makes it an ideal polyvalent vehicle for nanomachine fabrication, pathogen detection, and the delivery of therapeutics. This report describes novel approaches for the fabrication of polyvalent therapeutic pRNA nanoparticles, especially tetramers for specific siRNA delivery to cancer cells and for the silencing of targeted genes. RNA 3-D design, circular permutation, folding energy alteration, and nucleotide modification were applied to generate stable RNA nanoparticles with low toxicity. Animal trials demonstrated the high efficiency of the polyvalent RNA nanoparticles in the prevention and treatment of cancer. Using such protein-free nanoparticles as therapeutic reagents would allow for long-term administration to avoid the induction of antibody due to repeated treatment for chronic diseases.

Phi29 pRNA vector for efficient escort of hammerhead ribozyme targeting survivin in multiple cancer cells.

Ribozymes are potential therapeutic agents which suppress specific genes in disease-affected cells. Ribozymes have high substrate cleavage efficiency, yet their medical application has been hindered by RNA degradation, aberrant cell trafficking, or misfolding when fused to a carrier. In this study, we constructed a chimeric ribozyme escorted by the motor pRNA of bacteriophage phi29 to achieve proper folding and enhanced stability. A pRNA molecule contains an interlocking loop domain and a 5'/3' helical domain, which fold independently of one another. When a ribozyme is connected to the helical domain, the chimeric pRNA/ribozyme reorganizes into a circularly permuted form, and the 5'/3' ends are relocated and buried in the original 71'/75' positions. Effective silencing of the anti-apoptotic gene survivin by an appropriately designed chimeric ribozyme, as demonstrated at mRNA and protein levels, led to programmed cell death in various human cancer cell lines, including breast, prostate, cervical, nasopharyngeal, and lung, without causing significant non-specific cytotoxicity. Through the interlocking interaction of right and left loops, monomer pRNA/ribozyme chimeras can be incorporated into multi-functional dimer, trimer and hexamer complexes for specific gene delivery. Using the phi29 motor pRNA as an escort may revive the ribozyme's strength in medical application.