Reservoir of Fibroblasts Promotes Recovery From Limb Ischemia.

BACKGROUND: The angiogenic response to ischemia restores perfusion so as to preserve tissue. A role for mesenchymal-to-endothelial transition in the angiogenic response is controversial. This study is to determine if resident fibroblasts contribute to angiogenesis. METHODS: We utilized the murine model of hindlimb ischemia, and in vivo Matrigel plug assay together with lineage tracing studies and single cell RNA-sequencing to examine the transcriptional and functional changes in fibroblasts in response to ischemia. RESULTS: Lineage tracing using Fsp1-Cre: R26R-EYFP mice revealed the emergence within the ischemic hindlimb of a small subset of YFP+ CD144+ CD11b- fibroblasts (E* cells) that expressed endothelial cell (EC) genes. Subcutaneous administration of Matrigel in Fsp1-Cre: R26R-EYFP mice generated a plug that became vascularized within 5 days. Isolation of YFP+ CD11b- cells from the plug revealed a small subset of YFP+ CD144+ CD11b- E* cells which expressed EC genes. Pharmacological or genetic suppression of innate immune signaling reduced vascularity of the Matrigel plug and abrogated the generation of these E* cells. These studies were repeated using human fibroblasts, with fluorescence-activated cell sorting analysis revealing that a small percentage of human fibroblasts that were induced to express EC markers in Matrigel plug assay. Pharmacological suppression or genetic knockout of inflammatory signaling abolished the generation of E* cells, impaired perfusion recovery and increased tissue injury after femoral artery ligation. To further characterize these E* cells, single cell RNA-sequencing studies were performed and revealed 8 discrete clusters of cells expressing characteristic fibroblast genes, of which 2 clusters (C5 and C8) also expressed some EC genes. Ischemia of the hindlimb induced expansion of clusters C5 and C8. The C8 cells did not express CD144, nor did they form networks in Matrigel, but did generate angiogenic cytokines. The C5 fibroblasts most resembled E* cells in their expression of CD144 and their ability to form EC-like networks in Matrigel. CONCLUSIONS: Together, these studies indicate the presence of subsets of tissue fibroblasts which seem poised to contribute to the angiogenic response. The expansion of these subsets with ischemia is dependent on activation of innate immune signaling and contributes to recovery of perfusion and preservation of ischemic tissue.

Nuclear S-Nitrosylation Defines an Optimal Zone for Inducing Pluripotency.

BACKGROUND: We found that cell-autonomous innate immune signaling causes global changes in the expression of epigenetic modifiers to facilitate nuclear reprogramming to pluripotency. A role of S-nitrosylation by inducible nitric oxide (NO) synthase, an important effector of innate immunity, has been previously described in the transdifferentiation of fibroblasts to endothelial cells. Accordingly, we hypothesized that S-nitrosylation might also have a role in nuclear reprogramming to pluripotency. METHODS: We used murine embryonic fibroblasts containing a doxycycline-inducible cassette encoding the Yamanaka factors (Oct4, Sox2, Klf4, and c-Myc), and genetic or pharmacological inhibition of inducible NO synthase together with the Tandem Mass Tag approach, chromatin immunoprecipitation-quantitative polymerase chain reaction, site-directed mutagenesis, and micrococcal nuclease assay to determine the role of S-nitrosylation during nuclear reprogramming to pluripotency. RESULTS: We show that an optimal zone of innate immune activation, as defined by maximal yield of induced pluripotent stem cells, is determined by the degree of activation of nuclear factor κ-light-chain-enhancer of activated B cells; NO generation; S-nitrosylation of nuclear proteins; and DNA accessibility as reflected by histone markings and increased mononucleosome generation in a micrococcal nuclease assay. Genetic or pharmacological inhibition of inducible NO synthase reduces DNA accessibility and suppresses induced pluripotent stem cell generation. The effect of NO on DNA accessibility is mediated in part by S-nitrosylation of nuclear proteins, including MTA3 (Metastasis Associated 1 Family Member 3), a subunit of NuRD (Nucleosome Remodeling Deacetylase) complex. S-Nitrosylation of MTA3 is associated with decreased NuRD activity. Overexpression of mutant MTA3, in which the 2 cysteine residues are replaced by alanine residues, impairs the generation of induced pluripotent stem cells. CONCLUSIONS: This is the first report showing that DNA accessibility and induced pluripotent stem cell yield depend on the extent of cell-autonomous innate immune activation and NO generation. This "Goldilocks zone" for inflammatory signaling and epigenetic plasticity may have broader implications for cell fate and phenotypic fluidity.

Retinoic Acid Inducible Gene 1 Protein (RIG1)-Like Receptor Pathway Is Required for Efficient Nuclear Reprogramming.

We have revealed a critical role for innate immune signaling in nuclear reprogramming to pluripotency, and in the nuclear reprogramming required for somatic cell transdifferentiation. Activation of innate immune signaling causes global changes in the expression and activity of epigenetic modifiers to promote epigenetic plasticity. In our previous articles, we focused on the role of toll-like receptor 3 (TLR3) in this signaling pathway. Here, we define the role of another innate immunity pathway known to participate in response to viral RNA, the retinoic acid-inducible gene 1 receptor (RIG-1)-like receptor (RLR) pathway. This pathway is represented by the sensors of viral RNA, RIG-1, LGP2, and melanoma differentiation-associated protein 5 (MDA5). We first found that TLR3 deficiency only causes a partial inhibition of nuclear reprogramming to pluripotency in mouse tail-tip fibroblasts, which motivated us to determine the contribution of RLR. We found that knockdown of interferon beta promoter stimulator 1, the common adaptor protein for the RLR family, substantially reduced nuclear reprogramming induced by retroviral or by modified messenger RNA expression of Oct 4, Sox2, KLF4, and c-MYC (OSKM). Importantly, a double knockdown of both RLR and TLR3 pathway led to a further decrease in induced pluripotent stem cell (iPSC) colonies suggesting an additive effect of both these pathways on nuclear reprogramming. Furthermore, in murine embryonic fibroblasts expressing a doxycycline (dox)-inducible cassette of the genes encoding OSKM, an RLR agonist increased the yield of iPSCs. Similarly, the RLR agonist enhanced nuclear reprogramming by cell permeant peptides of the Yamanaka factors. Finally, in the dox-inducible system, RLR activation promotes activating histone marks in the promoter region of pluripotency genes. To conclude, innate immune signaling mediated by RLR plays a critical role in nuclear reprogramming. Manipulation of innate immune signaling may facilitate nuclear reprogramming to achieve pluripotency. Stem Cells 2017;35:1197-1207.

Transdifferentiation Requires iNOS Activation: Role of RING1A S-Nitrosylation.

RATIONALE: We have previously shown that innate immunity is necessary for transdifferentiation of fibroblasts to endothelial cells. A major signaling molecule involved in innate immunity is inducible nitric oxide synthase (iNOS). Accordingly, we hypothesized that iNOS-generated nitric oxide (NO) might enhance transdifferentiation. OBJECTIVE: To elucidate the role of NO in epigenetic plasticity during transdifferentiation. METHODS AND RESULTS: We exposed the BJ fibroblasts to transdifferentiation formulation that included endothelial growth factors and innate immune activator polyinosinic:polycytidylic acid to induce endothelial cells. Generation of transdifferentiated endothelial cells was associated with iNOS expression and NO elaboration. In the absence of polyinosinic:polycytidylic acid, or in the presence of antagonists of NFκB (nuclear factor kappa B) or iNOS activity, NO synthesis and induce endothelial cell generation was reduced. Furthermore, genetic knockout (in murine embryonic fibroblasts) or siRNA knockdown (in BJ fibroblasts) of iNOS nearly abolished transdifferentiation, an effect that could be reversed by iNOS overexpression. Notably, polyinosinic:polycytidylic acid induced nuclear localization of iNOS, and its binding to, and nitrosylation of, the epigenetic modifier ring finger protein 1A (RING1A) as assessed by immunostaining, Co-IP, and mass spectrometry. Nitrosylation of RING1A reduced its binding to chromatin and reduced global levels of repressive histone marker H3K27 trimethylation. Overexpression of a mutant form of RING1A (C398A) lacking the nitrosylation site almost abrogated transdifferentiation. CONCLUSIONS: Overall, our data indicate that during transdifferentiation, innate immune activation increases iNOS generation of NO to S-nitrosylate RING1A, a key member of the polycomb repressive complex. Nitrosylation of RING1A reduces its binding to chromatin and decreases H3K27 trimethylation level. The release of epigenetic repression by nitrosylation of RING1A is critical for effective transdifferentiation.

Green tissue-specific co-expression of chitinase and oxalate oxidase 4 genes in rice for enhanced resistance against sheath blight.

MAIN CONCLUSION: Green tissue-specific simultaneous overexpression of two defense-related genes ( OsCHI11 & OsOXO4 ) in rice leads to significant resistance against sheath blight pathogen ( R. solani ) without distressing any agronomically important traits. Overexpressing two defense-related genes (OsOXO4 and OsCHI11) cloned from rice is effective at enhancing resistance against sheath blight caused by Rhizoctonia solani. These genes were expressed under the control of two different green tissue-specific promoters, viz. maize phosphoenolpyruvate carboxylase gene promoter, PEPC, and rice cis-acting 544-bp DNA element, immediately upstream of the D54O translational start site, P D54O-544 . Putative T0 transgenic rice plants were screened by PCR and integration of genes was confirmed by Southern hybridization of progeny (T1) rice plants. Successful expression of OsOXO4 and OsCHI11 in all tested plants was confirmed. Expression of PR genes increased significantly following pathogen infection in overexpressing transgenic plants. Following infection, transgenic plants exhibited elevated hydrogen peroxide levels, significant changes in activity of ROS scavenging enzymes and reduced membrane damage when compared to their wild-type counterpart. In a Rhizoctonia solani toxin assay, a detached leaf inoculation test and an in vivo plant bioassay, transgenic plants showed a significant reduction in disease symptoms in comparison to non-transgenic control plants. This is the first report of overexpression of two different PR genes driven by two green tissue-specific promoters providing enhanced sheath blight resistance in transgenic rice.

ß-Lactams increase the antibacterial activity of daptomycin against clinical methicillin-resistant Staphylococcus aureus strains and prevent selection of daptomycin-resistant derivatives.

Methicillin-resistant Staphylococcus aureus (MRSA) has emerged to be one of the most important pathogens both in health care and in community-onset infections. Daptomycin (DAP) is a cyclic anionic lipopeptide recommended for treatment of skin infections, bacteremia, and right-sided endocarditis caused by MRSA. Resistance to DAP (DAP(r)) has been reported in MRSA and is mostly accompanied by a parallel decrease in oxacillin resistance, a process known as the "seesaw effect." Our study provides evidence that the seesaw effect applies to other ß-lactams and carbapenems of clinical use, including nafcillin (NAF), cefotaxime (CTX), amoxicillin-clavulanic (AMC), and imipenem (IMP), in heterogeneous DAP(r) MRSA strains but not in MRSA strains expressing homogeneous ß-lactam resistance. The antibacterial efficacy of DAP in combination with ß-lactams was evaluated in isogenic DAP-susceptible (DAP(s))/Dap(r) MRSA strains originally obtained from patients that failed DAP monotherapy. Both in vitro (MIC, synergy-kill curve) and in vivo (wax worm model) approaches were used. In these models, DAP and a ß-lactam proved to be highly synergistic against both heterogeneous and homogeneous clinical DAP(r) MRSA strains. Mechanistically, ß-lactams induced a reduction in the cell net positive surface charge, reverting the increased repulsion provoked by DAP alone, an effect that may favor the binding of DAP to the cell surface. The ease of in vitro mutant selection was observed when DAP(s) MRSA strains were exposed to DAP. Importantly, the combination of DAP and a ß-lactam prevented the selection of DAP(r) variants. In summary, our data show that the DAP-ß-lactam combination may significantly enhance both the in vitro and in vivo efficacy of anti-MRSA therapeutic options against DAP(r) MRSA infections and represent an option in preventing DAP(r) selection in persistent or refractory MRSA infections.

mRNA-Enhanced Cell Therapy and Cardiovascular Regeneration.

mRNA has emerged as an important biomolecule in the global call for the development of therapies during the COVID-19 pandemic. Synthetic in vitro-transcribed (IVT) mRNA can be engineered to mimic naturally occurring mRNA and can be used as a tool to target "undruggable" diseases. Recent advancement in the field of RNA therapeutics have addressed the challenges inherent to this drug molecule and this approach is now being applied to several therapeutic modalities, from cancer immunotherapy to vaccine development. In this review, we discussed the use of mRNA for stem cell generation or enhancement for the purpose of cardiovascular regeneration.

Characterization of an unusual cold shock protein from Staphylococcus aureus.

Of the three cold shock proteins expressed by Staphylococcus aureus, CspC is induced poorly by cold but strongly by various antibiotics and toxic chemicals. Using a purified CspC, here we demonstrate that it exists as a monomer in solution, possesses primarily ß-sheets, and bears substantial structural similarity with other bacterial Csps. Aggregation of CspC was initiated rapidly at temperatures above 40 °C, whereas, the Gibbs free energy of stabilization of CspC at 0 M GdmCl was estimated to be +1.6 kcal mol(-1), indicating a less stable protein. Surprisingly, CspC showed stable binding with ssDNA carrying a stretch of more than three thymine bases and binding with such ssDNA had not only stabilized CspC against proteolytic degradation but also quenched the fluorescence intensity from its exposed Trp residue. Analysis of quenching data indicates that each CspC molecule binds with ∼5 contiguous thymine bases of the above ssDNA and binding is cooperative in nature.

Cytoskeleton stiffness regulates cellular senescence and innate immune response in Hutchinson-Gilford Progeria Syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is caused by the accumulation of mutant prelamin A (progerin) in the nuclear lamina, resulting in increased nuclear stiffness and abnormal nuclear architecture. Nuclear mechanics are tightly coupled to cytoskeletal mechanics via lamin A/C. However, the role of cytoskeletal/nuclear mechanical properties in mediating cellular senescence and the relationship between cytoskeletal stiffness, nuclear abnormalities, and senescent phenotypes remain largely unknown. Here, using muscle-derived mesenchymal stromal/stem cells (MSCs) from the Zmpste24-/- (Z24-/- ) mouse (a model for HGPS) and human HGPS fibroblasts, we investigated the mechanical mechanism of progerin-induced cellular senescence, involving the role and interaction of mechanical sensors RhoA and Sun1/2 in regulating F-actin cytoskeleton stiffness, nuclear blebbing, micronuclei formation, and the innate immune response. We observed that increased cytoskeletal stiffness and RhoA activation in progeria cells were directly coupled with increased nuclear blebbing, Sun2 expression, and micronuclei-induced cGAS-Sting activation, part of the innate immune response. Expression of constitutively active RhoA promoted, while the inhibition of RhoA/ROCK reduced cytoskeletal stiffness, Sun2 expression, the innate immune response, and cellular senescence. Silencing of Sun2 expression by siRNA also repressed RhoA activation, cytoskeletal stiffness and cellular senescence. Treatment of Zmpste24-/- mice with a RhoA inhibitor repressed cellular senescence and improved muscle regeneration. These results reveal novel mechanical roles and correlation of cytoskeletal/nuclear stiffness, RhoA, Sun2, and the innate immune response in promoting aging and cellular senescence in HGPS progeria.

Antibiotics, arsenate and H2O2 induce the promoter of Staphylococcus aureus cspC gene more strongly than cold.

Proteins expressed by the bacterial cold shock genes are highly conserved at sequence level and perform various biological functions in both the cold-stressed and normal cells. To study the effects of various agents on the cold shock genes of Staphylococcus aureus, we have cloned the upstream region of cspC from S. aureus Newman and found that the above region possesses appreciable promoter (P(c)) activity even at 37 degrees C. A reporter S. aureus strain CHANDA2, constructed by inserting the P(c)-lacZ transcriptional fusion into S. aureus RN4220 genome, was found to express very low level of beta-galactosidase after cold shock, indicating that low temperature induces P(c) very weakly. Interestingly, transcription from P(c ) was induced very strongly by several antibiotics, hydrogen peroxide and arsenate salt. Cold shock proteins expressed by S. aureus are highly identical at sequence level and bear single-strand nucleic acid binding motifs. A 16 nt downstream box and a 13 nt upstream box were identified at the downstream of initiation codon and at the upstream of ribosome binding site of csp transcripts. Their roles in S. aureus cold shock gene expression have been discussed elaborately.

Transflammation: How Innate Immune Activation and Free Radicals Drive Nuclear Reprogramming.

SIGNIFICANCE: Yamanaka and colleagues galvanized the field of stem cell biology and regenerative medicine by their generation of induced pluripotent stem cells. Evidence is emerging that activation of innate immune signaling is critical for efficient reprogramming to pluripotency and for the nuclear reprogramming occurring in transdifferentiation. Recent Advances: We have shown that innate immune signaling triggers a global change in the expression of epigenetic modifiers to enhance DNA accessibility. In this state of epigenetic plasticity, overexpression of lineage determination factors, and/or environmental cues and paracrine factors, can induce pluripotency, or can direct transdifferentiation to another somatic cell lineage. Accumulating evidence reveals that innate immune activation triggers the generation of reactive oxygen species and reactive nitrogen species, and that these free radicals are required for nuclear reprogramming to pluripotency or for transdifferentiation. CRITICAL ISSUES: We have discovered a limb of innate immune signaling that regulates DNA accessibility, in part, by the action of free radicals to induce post-translational modification of epigenetic modifiers. FUTURE DIRECTIONS: It is of scientific interest and clinical relevance to understand the mechanisms by which free radicals influence epigenetic plasticity, and how these mechanisms may be therapeutically modulated. Antioxid. Redox Signal. 00, 000-000.

Repressor of temperate mycobacteriophage L1 harbors a stable C-terminal domain and binds to different asymmetric operator DNAs with variable affinity.

BACKGROUND: Lysogenic mode of life cycle of a temperate bacteriophage is generally maintained by a protein called 'repressor'. Repressor proteins of temperate lambdoid phages bind to a few symmetric operator DNAs in order to regulate their gene expression. In contrast, repressor molecules of temperate mycobacteriophages and some other phages bind to multiple asymmetric operator DNAs. Very little is known at present about the structure-function relationship of any mycobacteriophage repressor. RESULTS: Using highly purified repressor (CI) of temperate mycobacteriophage L1, we have demonstrated here that L1 CI harbors an N-terminal domain (NTD) and a C-terminal domain (CTD) which are separated by a small hinge region. Interestingly, CTD is more compact than NTD at 25 degrees C. Both CTD and CI contain significant amount of alpha-helix at 30 degrees C but unfold partly at 42 degrees C. At nearly 200 nM concentration, both proteins form appreciable amount of dimers in solution. Additional studies reveal that CI binds to O64 and OL types of asymmetric operators of L1 with variable affinity at 25 degrees C. Interestingly, repressor-operator interaction is affected drastically at 42 degrees C. The conformational change of CI is most possibly responsible for its reduced operator binding affinity at 42 degrees C. CONCLUSION: Repressors encoded by mycobacteriophages differ significantly from the repressor proteins of lambda and related phages at functional level but at structural level they are nearly similar.

Detection of antistaphylococcal and toxic compounds by biological assay systems developed with a reporter Staphylococcus aureus strain harboring a heat inducible promoter - lacZ transcriptional fusion.

Previously it was reported that promoter of groES-groEL operon of Staphylococcus aureus is induced by various cell-wall active antibiotics. In order to exploit the above promoter for identifying novel antistaphylococcal drugs, we have cloned the promoter containing region (P(g)) of groES-groEL operon of S. aureus Newman and found that the above promoter is induced by sublethal concentrations of many antibiotics including cell-wall active antibiotics. A reporter S. aureus RN4220 strain (designated SAU006) was constructed by inserting the P(g)-lacZ transcriptional fusion into its chromosome. Agarose-based assay developed with SAU006 shows that P(g) in single-copy is also induced distinctly by different classes of antibiotics. Data indicate that ciprofloxacin, rifampicin, ampicillin, and cephalothin are strong inducers, whereas, tetracycline, streptomycin and vancomycin induce the above promoter weakly. Sublethal concentrations of ciprofloxacin and ampicilin even have induced P(g) efficiently in microtiter plate grown SAU006. Additional studies show for the first time that above promoter is also induced weakly by arsenate salt and hydrogen peroxide. Taken together, we suggest that our simple and sensitive assay systems with SAU006 could be utilized for screening and detecting not only novel antistaphylococcal compounds but also different toxic chemicals.

Purification and characterization of repressor of temperate S. aureus phage phi11.

To gain insight into the structure and function of repressor proteins of bacteriophages of gram-positive bacteria, repressor of temperate Staphylococcus aureus phage phi11 was undertaken as a model system here and purified as an N-terminal histidine-tagged variant (His-CI) by affinity chromatography. A approximately 19 kDa protein copurified with intact His-CI (approximately 30 kDa) at low level was resulted most possibly due to partial cleavage at its Ala-Gly site. At approximately 10 nM and higher concentrations, His-CI forms significant amount of dimers in solution. There are two repressor binding sites in phi11 cI-cro intergenic region and binding to two sites occurs possibly by a cooperative manner. Two sites dissected by HincII digestion were designated operators O(L) and O(R), respectively. Equilibrium binding studies indicate that His-CI binds to O(R) with a little more strongly than O(L) and binding species is probably dimeric in nature. Interestingly His-CI binding affinity reduces drastically at elevated temperatures (32-42 degrees C). Both O(L) and O(R) harbor a nearly identical inverted repeat and studies show that phi11 repressor binds to each repeat efficiently. Additional analyses indicate that phi11 repressor, like lambda repressor, harbors an N-terminal domain and a C-terminal domain which are separated by a hinge region. Secondary structure of phi11 CI even nearly resembles to that of lambda, phage repressor though they differ at sequence level. The putative N-terminal HTH (helix-turn-helix) motif of phi11 repressor belongs to the HTH -XRE-family of proteins and shows significant identity to the HTH motifs of some proteins of evolutionary distant organisms but not to HTH motifs of most S. aureus phage repressors.

Transflammation: Innate immune signaling in nuclear reprogramming.

Induction of pluripotency in somatic cells by retroviral overexpression of four transcription factors has revolutionized the field of stem cell biology and regenerative medicine. The efficient induction of pluripotency requires the activation of innate immune signaling in a process termed "transflammation" (Lee et al., 2012). Specifically, the stimulation of pattern recognition receptors (PRRs) causes global alterations in the expression and activity of epigenetic modifiers to favor an open chromatin configuration. Activation of toll-like receptors (TLR) or RIG-1-like receptors (RLR) (Sayed et al. 2017) trigger signaling cascades that result in NFκB or IRF-3 mediated changes in epigenetic plasticity that facilitate reprogramming. Another form of nuclear reprogramming is so-called direct reprogramming or transdifferentiation of one somatic cell to another lineage. We have shown that transdifferentiation of human fibroblasts to endothelial cells also involves transflammation (Sayed et al., 2015). Recently, we also identified reactive oxygen species (ROS) (Zhou et al. 2016) and reactive nitrogen species (RNS) (Meng et al., 2016) as mediators of innate immune signaling in nuclear reprogramming. Innate immune signaling plays a key role in nuclear reprogramming by regulating DNA accessibility (Fig. 1). Here, we review recent progress of innate immunity signaling in nuclear reprogramming and epigenetic plasticity.

Effects of physical, ionic, and structural factors on the binding of repressor of mycobacteriophage L1 to its cognate operator DNA.

To determine the factors influencing the binding of L1 repressor to its cognate operator DNA, several gel shift as well as bioinformatic analyses have been carried out. The data show that time, temperature, salt, and pH each greatly affect the binding. In order to achieve optimum operator binding of L1 repressor in Tris buffer, the minimum requirements of time, temperature, salt, and pH were estimated to be 1 min, 32 degrees C, NaCl (50 mM), and 7.9, respectively. Interestingly Na+ but not NH4+, K+, or Li+ was found to augment significantly the binding activity of CI protein above the basal level. Anions like Cl-, citrate-, acetate-, and H2PO4- do not alter the binding of L1 repressor to its operator. We also show that an in frame deletion mutant of L1 repressor which does not carry the putative HTH motif (at its N-terminal end) fails to bind to its cognate operator DNA even at very high concentrations. The putative HTH motif was found highly conserved and evolutionarily very close to that of regulatory proteins of Y. pestis, H. marismortui, A. tumefaciens, etc. Taken together we suggest that N-terminal end of L1 repressor carries a HTH motif. Further analysis of the putative secondary structures of mycobacteriophage repressors reveals that two common regions encompassing more than 90% of primary sequence are present in all the four repressor molecules studied here. The results suggest that these common regions are utilized for carrying out identical functions.

Tissue-specific expression of Arabidopsis NPR1 gene in rice for sheath blight resistance without compromising phenotypic cost.

Rice sheath blight disease, caused by the fungus Rhizoctonia solani, is considered the second most important disease of rice after blast. NPR1 (non expressor of PR1) is the central regulator of systemic acquired resistance (SAR) conferring broad spectrum resistance to various pathogens. Previous reports have indicated that constitutive expression of the Arabidopsis thaliana NPR1 (AtNPR1) gene results in disease resistance in rice but has a negative impact on growth and agronomic traits. Here, we report that green tissue-specific expression of AtNPR1 in rice confers resistance to the sheath blight pathogen, with no concomitant abnormalities in plant growth and yield parameters. Elevated levels of NPR1 activated the defence pathway in the transgenic plants by inducing expression of endogenous genes such as PR1b, RC24, and PR10A. Enhanced sheath blight resistance of the transgenic plants was evaluated using three different bioassay systems. A partially isolated toxin from R. solani was used in the bioassays to measure the resistance level. Studies of the phenotype and yield showed that the transgenic plants did not exhibit any kind of phenotypic imbalances. Our results demonstrate that green tissue-specific expression of AtNPR1 is an effective strategy for controlling the sheath blight pathogen. The present work in rice can be extended to other crop plants severely damaged by the pathogen.

A point mutation at the C-terminal half of the repressor of temperate mycobacteriophage L1 affects its binding to the operator DNA.

The wild-type repressor CI of temperate mycobacteriophage L1 and the temperature-sensitive (ts) repressor CIts391 of a mutant L1 phage, L1cIts391, have been separately overexpressed in E. coli. Both these repressors were observed to specifically bind with the same cognate operator DNA. The operator-binding activity of CIts391 was shown to differ significantly than that of the CI at 32 to 42 degrees C. While 40-95% operator-binding activity was shown to be retained at 35 to 42 degrees C in CI, more than 75% operator-binding activity was lost in CIts391 at 35 to 38 degrees C, although the latter showed only 10% less binding compared to that of the former at 32 degrees C. The CIts391 showed almost no binding at 42 degrees C. An in vivo study showed that the CI repressor inhibited the growth of a clear plaque former mutant of the L1 phage more strongly than that of the CIts391 repressor at both 32 and 42 degrees C. The half-life of the CIts391-operator complex was found to be about 8 times less than that of the CI-operator complex at 32 degrees C. Interestingly, the repressor-operator complexes preformed at 0 degrees C have shown varying degrees of resistance to dissociation at the temperatures which inhibit the formation of these complexes are inhibited. The CI repressor, but not that of CIts391, regains most of the DNA-binding activity on cooling to 32 degrees C after preincubation at 42 to 52 degrees C. All these data suggest that the 131(st) proline residue at the C-terminal half of CI, which changed to leucine in the CIts391, plays a crucial role in binding the L1 repressor to the cognate operator DNA, although the helix-turn-helix DNA-binding motif of the L1 repressor is located at its N-terminal end.

Rice oxalate oxidase gene driven by green tissue-specific promoter increases tolerance to sheath blight pathogen (Rhizoctonia solani) in transgenic rice.

Rice sheath blight, caused by the necrotrophic fungus Rhizoctonia solani, is one of the most devastating and intractable diseases of rice, leading to a significant reduction in rice productivity worldwide. In this article, in order to examine sheath blight resistance, we report the generation of transgenic rice lines overexpressing the rice oxalate oxidase 4 (Osoxo4) gene in a green tissue-specific manner which breaks down oxalic acid (OA), the pathogenesis factor secreted by R. solani. Transgenic plants showed higher enzyme activity of oxalate oxidase (OxO) than nontransgenic control plants, which was visualized by histochemical assays and sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Transgenic rice leaves were more tolerant than control rice leaves to exogenous OA. Transgenic plants showed a higher level of expression of other defence-related genes in response to pathogen infection. More importantly, transgenic plants exhibited significantly enhanced durable resistance to R. solani. The overexpression of Osoxo4 in rice did not show any detrimental phenotypic or agronomic effect. Our findings indicate that rice OxO can be utilized effectively in plant genetic manipulation for sheath blight resistance, and possibly for resistance to other diseases caused by necrotrophic fungi, especially those that secrete OA. This is the first report of the expression of defence genes in rice in a green tissue-specific manner for sheath blight resistance.