Trans-complementation of the viral movement protein mediates efficient expression of large target genes via a tobacco mosaic virus vector.

The development of plant virus-based expression systems has expanded rapidly owing to their potential applications in gene functional and disease resistance research, and industrial production of pharmaceutical proteins. However, the low yield of certain proteins, especially high-molecular-mass proteins, restricts the production scale. In this study, we observed that the tobacco mosaic virus (TMV)-mediated expression of a foreign protein was correlated with the amount of the movement protein (MP) and developed a TMV-derived pAT-transMP vector system incorporating trans-complementation expression of MP. The system is capable of efficient expression of exogenous proteins, in particular those with a high molecular mass, and enables simultaneous expression of two target molecules. Furthermore, viral expression of competent CRISPR-Cas9 protein and construction of CRISPR-Cas9-mediated gene-editing system in a single pAT-transMP construct was achieved. The results demonstrated a novel role for TMV-MP in enhancing the accumulation of a foreign protein produced from the viral vector or a binary expression system. Further investigation of the mechanism underlying this role will be beneficial for optimization of plant viral vectors with broad applications.

Red-light is an environmental effector for mutualism between begomovirus and its vector whitefly.

Environments such as light condition influence the spread of infectious diseases by affecting insect vector behavior. However, whether and how light affects the host defense which further affects insect preference and performance, remains unclear, nor has been demonstrated how pathogens co-adapt light condition to facilitate vector transmission. We previously showed that begomoviral ßC1 inhibits MYC2-mediated jasmonate signaling to establish plant-dependent mutualism with its insect vector. Here we show red-light as an environmental catalyzer to promote mutualism of whitefly-begomovirus by stabilizing ßC1, which interacts with PHYTOCHROME-INTERACTING FACTORS (PIFs) transcription factors. PIFs positively control plant defenses against whitefly by directly binding to the promoter of terpene synthase genes and promoting their transcription. Moreover, PIFs interact with MYC2 to integrate light and jasmonate signaling and regulate the transcription of terpene synthase genes. However, begomovirus encoded ßC1 inhibits PIFs' and MYC2' transcriptional activity via disturbing their dimerization, thereby impairing plant defenses against whitefly-transmitted begomoviruses. Our results thus describe how a viral pathogen hijacks host external and internal signaling to enhance the mutualistic relationship with its insect vector.

Type III Secretion System Repressor RhpR Induces GrlP, a Glycine-Rich Outer Membrane Lipoprotein with Functions in Regulating the Periplasmic Space and Pleiotropic Responses.

The two-component system RhpRS was initially identified as a regulator of genes encoding the type III secretion system (T3SS) in Pseudomonas syringae. Phosphorylated RhpR (P-RhpR) negatively regulates the T3SS genes by repressing the hrpR promoter, but directly activates the expression of a small gene named here as grlp. Here, we show that grlp is expressed higher in rich medium than in minimal medium in P. s. pv. tomato DC3000 and encodes a glycine rich lipoprotein (GrlP) located in the outer membrane (OM). The grlp gene has a pleiotropic effect on bacterial behaviors such as reductions in pathogenicity, swimming motility, biofilm formation, tolerance to various stresses and antibiotics, and long-term survival when overexpressed, but induces these responses when it is deleted in P. s. pv. tomato DC3000. Overexpression of grlp increases the size of periplasm while deletion of grlp decreases the periplasmic space. Further, GrlP interacts with OprI, the ortholog of E. coli OM lipoprotein Lpp, a key player in determining the size of periplasm and mechanic stiffness of the OM by tethering the OM to peptidoglycan (PG) in periplasm. As periplasmic space and OM mechanics play central roles in regulating bacterial physiology, we speculate that GrlP probably imposes its functions on bacterial physiology by regulating the periplasmic space and OM mechanics. These findings suggest that the T3SS gene regulation is closely coordinated with bacterial cell envelope properties by RhpRS in P. syringe. IMPORTANCE The OM of Gram-negative bacteria is the most front line in contact with extracellular milieu. OM is not only a protective layer, but also a structure that determines the envelope stiffness. Recent evidence indicated that components determining the periplasmic space and cross-links of lipopolysaccharide on the OM play key roles in regulating the mechanical properties of the OM. However, whether the OM composition and mechanical properties are coordinated with the expression of the T3SS genes is unknown. Here, we found that the two-component system (TCS) regulator P-RhpR, a direct repressor of the T3SS regulator hrpRS operon, directly activates the expression of the OM lipoprotein gene grlp bearing a function in regulating the periplasmic space. This finding suggests a coordination between the OM properties and the T3SS gene regulation and reveals a new target for control of the T3SS gene expression and bacterial pathogenicity.

Structure-function analyses of coiled-coil immune receptors define a hydrophobic module for improving plant virus resistance.

Plant immunity relies on nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) that detect microbial patterns released by pathogens, and activate localized cell death to prevent the spread of pathogens. Tsw is the only identified resistance (R) gene encoding an NLR, conferring resistance to tomato spotted wilt orthotospovirus (TSWV) in pepper species (Capsicum, Solanaceae). However, molecular and cellular mechanisms of Tsw-mediated resistance are still elusive. Here, we analysed the structural and cellular functional features of Tsw protein, and defined a hydrophobic module to improve NLR-mediated virus resistance. The plasma membrane associated N-terminal 137 amino acid in the coiled-coil (CC) domain of Tsw is the minimum fragment sufficient to trigger cell death in Nicotiana benthamiana plants. Transient and transgenic expression assays in plants indicated that the amino acids of the hydrophobic groove (134th-137th amino acid) in the CC domain is critical for its full function and can be modified for enhanced disease resistance. Based on the structural features of Tsw, a super-hydrophobic funnel-like mutant, TswY137W, was identified to confer higher resistance to TSWV in a SGT1 (Suppressor of G-two allele of Skp1)-dependent manner. The same point mutation in a tomato Tsw-like NLR protein also improved resistance to pathogens, suggesting a feasible way of structure-assisted improvement of NLRs.

Heterosis in root microbiota inhibits growth of soil-borne fungal pathogens in hybrid rice.

In nature, plants are colonized by various microbes that play essential roles in their growth and health. Heterosis is a natural genetic phenomenon whereby first-generation hybrids exhibit superior phenotypic performance relative to their parents. It remains unclear whether this concept can be extended to the "hybridization" of microbiota from two parents in their descendants and what benefits the hybrid microbiota might convey. Here, we investigated the structure and function of the root microbiota from three hybrid rice varieties and their parents through amplicon sequencing analysis of bacterial 16S ribosomal DNA (rDNA) and fungal internal transcribed spacer (ITS) regions. We show that the bacterial and fungal root microbiota of the varieties are distinct from those of their parental lines and exhibit potential heterosis features in diversity and composition. Moreover, the root bacterial microbiota of hybrid variety LYP9 protects rice against soil-borne fungal pathogens. Systematic analysis of the protective capabilities of individual strains from a 30-member bacterial synthetic community derived from LYP9 roots indicated that community members have additive protective roles. Global transcription profiling analyses suggested that LYP9 root bacterial microbiota activate rice reactive oxygen species production and cell wall biogenesis, contributing to heterosis for protection. In addition, we demonstrate that the protection conferred by the LYP9 root microbiota is transferable to neighboring plants, potentially explaining the observed hybrid-mediated superior effects of mixed planting. Our findings suggest that some hybrids exhibit heterosis in their microbiota composition that promotes plant health, highlighting the potential for microbiota heterosis in breeding hybrid crops.

GrpE is involved in mitochondrial function and is an effective target for RNAi-mediated pest and arbovirus control.

Laodelphax striatellus is a sap-feeding pest and the main insect vector of rice stripe virus (RSV). There is an urgent need to identify molecular targets to control this insect pest and plant arboviruses. In this study, we identified a L. striatellus gene (named LsGrpE) encoding a GroP-E-like protein. We found that the LsGrpE protein localized to mitochondria. Using gene-specific dsRNA to interfere with the expression of LsGrpE led to a significant increase in insect mortality, and most of the surviving insects could not develop into adults. Further analyses revealed that LsGrpE deficiency caused mitochondrial dysfunction and inhibited the insulin pathway, resulting in diabetes-like symptoms such as elevated blood sugar, inactive behaviour, developmental delay, and death. In addition, LsGrpE deficiency significantly reduced the RSV titre in surviving L. striatellus, and indirectly prevented viral vertical transmission by reducing the number of adults. We generated transgenic rice plants expressing LsGrpE-specific dsRNA, and the dsRNA was acquired by L. striatellus during feeding, resulting in increased insect mortality and the prevention of arboviral transmission. This study clarifies the function of LsGrpE and demonstrates that LsGrpE can be used as a molecular target of plant-generated dsRNA to resist this sap-feeding pest, a17nd therefore, its transmitted arboviruses.

The histone deacetylase HDA703 interacts with OsBZR1 to regulate rice brassinosteroid signaling, growth and heading date through repression of Ghd7 expression.

The plant steroid hormones brassinosteroids (BRs) play crucial roles in plant growth and development. The BR signal transduction pathway from perception to the key transcription factors has been well understood in Arabidopsis thaliana and in rice (Oryza sativa); however, the mechanisms conferring BR-mediated growth and flowering remain largely unknown, especially in rice. In this study, we show that HDA703 is a histone H4K8 and H4K12 deacetylase in rice. Hda703 mutants display a typical BR loss-of-function phenotype and reduced sensitivity to brassinolide, the most active BR. Rice plants overexpressing HDA703 exhibit some BR gain-of-function phenotypes dependent on BR biosynthesis and signaling. We also show that HDA703 is a direct target of BRASSINAZOLE-RESISTANT1 (OsBZR1), a primary regulator of rice BR signaling, and HDA703 interacts with OsBZR1 in rice. We further show that GRAIN NUMBER, PLANT HEIGHT, and HEADING DATE 7 (Ghd7), a central regulator of growth, development, and the stress response, is a direct target of OsBZR1. HDA703 directly targets Ghd7 and represses its expression through histone H4 deacetylation. HDA703-overexpressing rice plants phenocopy Ghd7-silencing rice plants in both growth and heading date. Together, our study suggests that HDA703, a histone H4 deacetylase, interacts with OsBZR1 to regulate rice BR signaling, growth, and heading date through epigenetic regulation of Ghd7.

The Orthotospovirus nonstructural protein NSs suppresses plant MYC-regulated jasmonate signaling leading to enhanced vector attraction and performance.

Pandemics of vector-borne human and plant diseases often depend on the behaviors of their arthropod vectors. Arboviruses, including many bunyaviruses, manipulate vector behavior to accelerate their own transmission to vertebrates, birds, insects, and plants. However, the molecular mechanism underlying this manipulation remains elusive. Here, we report that the non-structural protein NSs of Tomato spotted wilt orthotospovirus, a prototype of the Tospoviridae family and the Orthotospovirus genus, is a key viral factor that indirectly modifies vector preference and increases vector performance. NSs suppresses the biosynthesis of plant volatile monoterpenes, which serve as repellents of the vector western flower thrips (WFT, Frankliniella occidentalis). NSs directly interacts with MYC2, the jasmonate (JA) signaling master regulator and its two close homologs MYC3 and MYC4, to disable JA-mediated activation of terpene synthase genes. The dysfunction of the MYCs subsequently attenuates host defenses, increases the attraction of thrips, and improves thrips fitness. Moreover, MYC2 associated with NSs of Tomato zonate spot orthotospovirus, another Euro/Asian-type orthotospovirus, suggesting that MYC2 is an evolutionarily conserved target of Orthotospovirus species for suppression of terpene-based resistance to promote vector performance. These findings elucidate the molecular mechanism through which an orthotospovirus indirectly manipulates vector behaviors and therefore facilitates pathogen transmission. Our results provide insights into the molecular mechanisms by which Orthotospovirus NSs counteracts plant immunity for pathogen transmission.

Two components of the rhpPC operon coordinately regulate the type III secretion system and bacterial fitness in Pseudomonas savastanoi pv. phaseolicola.

Many plant bacterial pathogens including Pseudomonas species, utilize the type III secretion system (T3SS) to deliver effector proteins into plant cells. Genes encoding the T3SS and its effectors are repressed in nutrient-rich media but are rapidly induced after the bacteria enter a plant or are transferred into nutrient-deficient media. To understand how the T3SS genes are regulated, we screened for P. savastanoi pv. phaseolicola (Psph) mutants displaying diminished induction of avrPto-luc, a reporter for the T3SS genes, in Arabidopsis. A mutant carrying transposon insertion into a gene coding for a small functional unknown protein, designated as rhpC, was identified that poorly induced avrPto-luc in plants and in minimal medium (MM). Interestingly, rhpC is located immediately downstream of a putative metalloprotease gene named rhpP, and the two genes are organized in an operon rhpPC; but rhpP and rhpC displayed different RNA expression patterns in nutrient-rich King's B medium (KB) and MM. Deletion of the whole rhpPC locus did not significantly affect the avrPto-luc induction, implying coordinated actions of rhpP and rhpC in regulating the T3SS genes. Further analysis showed that RhpC was a cytoplasmic protein that interacted with RhpP and targeted RhpP to the periplasm. In the absence of RhpC, RhpP was localized in the cytoplasm and caused a reduction of HrpL, a key regulator of the T3SS genes, and also reduced the fitness of Psph. Expression of RhpP alone in E. coli inhibited the bacterial growth. The detrimental effect of RhpP on the fitness of Psph and E. coli required metalloprotease active sites, and was repressed when RhpC was co-expressed with RhpP. The coordination between rhpP and rhpC in tuning the T3SS gene expression and cell fitness reveals a novel regulatory mechanism for bacterial pathogenesis. The function of RhpP in the periplasm remains to be studied.

Rice GERMIN-LIKE PROTEIN 2-1 Functions in Seed Dormancy under the Control of Abscisic Acid and Gibberellic Acid Signaling Pathways.

Seed dormancy is a natural phenomenon in plants. It ensures that seeds complete the grain-filling stage before germination and prevents germination in unsuitable ecological conditions. In this study, we determined the previously unknown function of the rice (Oryza sativa) gene GERMIN-LIKE PROTEIN 2-1 (OsGLP2-1) in seed dormancy. Using artificial microRNA and CRISPR/CAS9 approaches, suppression of OsGLP2-1 expression in rice resulted in the release of dormancy in immature seeds. Conversely, overexpression of OsGLP2-1 driven by the OsGLP2-1 native promoter led to greater seed dormancy. Seed scutellum-specific expression of OsGLP2-1 was increased by exogenous abscisic acid, but decreased with gibberellic acid treatment. We provide evidence that OsGLP2-1 is antagonistically controlled at the transcriptional level by ABA INSENSITIVE5 and GAMYB transcription factors. We conclude that OsGLP2-1 acts as a buffer, maintaining appropriate equilibrium for the regulation of primary dormancy during seed development in rice.

Promotion of BR Biosynthesis by miR444 Is Required for Ammonium-Triggered Inhibition of Root Growth.

Rice (Oryza sativa), the staple food for almost half of the world's population, prefers ammonium (NH4 +) as the major nitrogen resource, and while NH4 + has profound effects on rice growth and yields, the underlying regulatory mechanisms remain largely unknown. Brassinosteroids (BRs) are a class of steroidal hormones playing key roles in plant growth and development. In this study, we show that NH4 + promotes BR biosynthesis through miR444 to regulate rice root growth. miR444 targeted five homologous MADS-box transcription repressors potentially forming homologous or heterogeneous complexes in rice. miR444 positively regulated BR biosynthesis through its MADS-box targets, which directly repress the transcription of BR-deficient dwarf 1 (OsBRD1), a key BR biosynthetic gene. NH4 + induced the miR444-OsBRD1 signaling cascade in roots, thereby increasing the amount of BRs, whose biosynthesis and signaling were required for NH4 + -dependent root elongation inhibition. Consistently, miR444-overexpressing rice roots were hypersensitive to NH4 + depending on BR biosynthesis, and overexpression of miR444's target, OsMADS57, resulted in rice hyposensitivity to NH4 + in root elongation, which was associated with a reduction of BR content. In summary, our findings reveal a cross talk mechanism between NH4 + and BR in which NH4 + activates miR444-OsBRD1, an undescribed BR biosynthesis-promoting signaling cascade, to increase BR content, inhibiting root elongation in rice.

A 7-Amino-Acid Motif of Rep Protein Essential for Virulence Is Critical for Triggering Host Defense Against Sri Lankan Cassava Mosaic Virus.

Geminiviruses cause severe damage to agriculture worldwide. The replication (Rep) protein is the indispensable viral protein for viral replication. Although various functional domains of Rep protein in Geminivirus spp. have been characterized, the most carboxyl terminus of Rep protein was not available. We have reported the first cassava-infecting geminivirus, Sri Lankan cassava mosaic virus (SLCMV-HN7 strain), in China. In this study, we reported the second Chinese SLCMV strain, SLCMV-Col, and conducted comparative genomic analysis between these two SLCMV strains. The virulence of SLCMV-Col is much stronger than SLCMV-HN7, indicated by the higher virus titer, more severe symptoms, and more extent host defense. We functionally characterized that Rep protein, a 7-amino-acid motif at the most carboxyl terminus, is essential for Rep protein accumulation and virulence of SLCMV. We also provided evidence suggesting that the motif could also enhance triggering of salicylic acid (SA) defense against SLCMV infection in Nicotiana benthamiana. The significance of the balance between virulence and host SA defense responses in expanding invasions of SLCMV is also discussed.

Insect tissue-specific vitellogenin facilitates transmission of plant virus.

Insect vitellogenin (Vg) has been considered to be synthesized in the fat body. Here, we found that abundant Vg protein is synthesized in Laodelphax striatellus hemocytes as well. We also determined that only the hemocyte-produced Vg binds to Rice stripe virus (RSV) in vivo. Examination of the subunit composition of L. striatellus Vg (LsVg) revealed that LsVg was processed differently after its expression in different tissues. The LsVg subunit able to bind to RSV exist stably only in hemocytes, while fat body-produced LsVg lacks the RSV-interacting subunit. Nymph and male L. striatellus individuals also synthesize Vg but only in hemocytes, and the proteins co-localize with RSV. We observed that knockdown of LsVg transcripts by RNA interference decreased the RSV titer in the hemolymph, and thus interfered with systemic virus infection. Our results reveal the sex-independent expression and tissue-specific processing of LsVg and also unprecedentedly connect the function of this protein in mediating virus transmission to its particular molecular forms existing in tissues previously known as non-Vg producing.

Phytochrome-interacting factor-like protein OsPIL15 integrates light and gravitropism to regulate tiller angle in rice.

MAIN CONCLUSION: Rice phytochrome-interacting factor-like protein OsPIL15 regulates tiller angle through light and gravity signals in rice. Tiller angle of cereal crops is a key agronomic trait that contributes to grain production. An understanding of how tiller angle is controlled is helpful for achieving ideal plant architecture to improve grain yield. Phytochrome-interacting factors (PIFs) are known to regulate seed germination, seedling skotomorphogenesis, shade avoidance, and flowering in Arabidopsis thaliana. Here, we report that OsPIL15 is, indeed, a rice PIF that negatively regulates tiller angle. Dominant-negative OsPIL15 plants displayed a larger tiller angle, which was associated with reduced shoot gravitropism. Phytochrome B (phyB) is the main photoreceptor perceiving the low red:far-red ratio of shade light. Compared with wild-type rice plants, loss-of-function phyB plants and OsPIL15-overexpressing plants showed smaller tiller angles and enhanced shoot gravitropism. In addition, more OsPIL15 protein accumulated in phyB plants than in wild-type plants. Light regulates the level of the OsPIL15 protein negatively, depending on phyB partially. We propose that OsPIL15 integrates light and gravity signals to regulate tiller angle in rice.

Geminivirus Activates ASYMMETRIC LEAVES 2 to Accelerate Cytoplasmic DCP2-Mediated mRNA Turnover and Weakens RNA Silencing in Arabidopsis.

Aberrant viral RNAs produced in infected plant cells serve as templates for the synthesis of dsRNAs. The derived virus-related small interfering RNAs (siRNA) mediate cleavage of viral RNAs by post-transcriptional gene silencing (PTGS), thus blocking virus multiplication. Here, we identified ASYMMETRIC LEAVES2 (AS2) as a new component of plant P body complex which mediates mRNA decapping and degradation. We found that AS2 promotes DCP2 decapping activity, accelerates mRNA turnover rate, inhibits siRNA accumulation and functions as an endogenous suppressor of PTGS. Consistent with these findings, as2 mutant plants are resistant to virus infection whereas AS2 over-expression plants are hypersensitive. The geminivirus nuclear shuttle protein BV1 protein, which shuttles between nuclei and cytoplasm, induces AS2 expression, causes nuclear exit of AS2 to activate DCP2 decapping activity and renders infected plants more sensitive to viruses. These principles of gene induction and shuttling of induced proteins to promote mRNA decapping in the cytosol may be used by viral pathogens to weaken antiviral defenses in host plants.

A Signaling Cascade from miR444 to RDR1 in Rice Antiviral RNA Silencing Pathway.

Plant RNA-DEPENDENT RNA POLYMERASE1 (RDR1) is a key component of the antiviral RNA-silencing pathway, contributing to the biogenesis of virus-derived small interfering RNAs. This enzyme also is responsible for producing virus-activated endogenous small interfering RNAs to stimulate the broad-spectrum antiviral activity through silencing host genes. The expression of RDR1 orthologs in various plants is usually induced by virus infection. However, the molecular mechanisms of activation of RDR1 expression in response to virus infection remain unknown. Here, we show that a monocot-specific microRNA, miR444, is a key factor in relaying the antiviral signaling from virus infection to OsRDR1 expression. The expression of miR444 is enhanced by infection with Rice stripe virus (RSV), and overexpression of miR444 improves rice (Oryza sativa) resistance against RSV infection accompanied by the up-regulation of OsRDR1 expression. We further show that three miR444 targets, the MIKC(C)-type MADS box proteins OsMADS23, OsMADS27a, and OsMADS57, form homodimers and heterodimers between them to repress the expression of OsRDR1 by directly binding to the CArG motifs of its promoter. Consequently, an increased level of miR444 diminishes the repressive roles of OsMADS23, OsMADS27a, and OsMADS57 on OsRDR1 transcription, thus activating the OsRDR1-dependent antiviral RNA-silencing pathway. We also show that overexpression of miR444-resistant OsMADS57 reduced OsRDR1 expression and rice resistance against RSV infection, and knockout of OsRDR1 reduced rice resistance against RSV infection. In conclusion, our results reveal a molecular cascade in the rice antiviral pathway in which miR444 and its MADS box targets directly control OsRDR1 transcription.

Transovarial transmission of a plant virus is mediated by vitellogenin of its insect vector.

Most plant viruses are transmitted by hemipteroid insects. Some viruses can be transmitted from female parent to offspring usually through eggs, but the mechanism of this transovarial transmission remains unclear. Rice stripe virus (RSV), a Tenuivirus, transmitted mainly by the small brown planthopper (Laodelphax striatellus), is also spread to the offspring through the eggs. Here, we used the RSV-planthopper system as a model to investigate the mechanism of transovarial transmission and demonstrated the central role of vitellogenin (Vg) of L. striatellus in the process of virus transmission into the eggs. Our data showed Vg can bind to pc3 in vivo and in vitro and colocalize in the germarium. RSV filamentous ribonucleoprotein particles (RNPs) only accumulated in the terminal filaments and pedicel areas prior to Vg expression and was not present in the germarium until Vg was expressed, where RSV RNPs and Vg had colocalized. Observations by immunoelectron microscopy (IEM) also indicated that these two proteins colocalized in nurse cells. Knockdown of Vg expression due to RNA interference resulted in inhibition of the invasion of ovarioles by RSV. Together, the data obtained indicated that RSV RNPs may enter the nurse cell of the germarium via endocytosis through binding with Vg. Finally, the virus enters the oocytes through nutritive cords, using the same route as for Vg transport. Our results show that the Vg of L. striatellus played a critical role in transovarial transmission of RSV and shows how viruses can use existing transovarial transportation systems in insect vectors for their own purposes.

miR444a has multiple functions in the rice nitrate-signaling pathway.

Nitrate (NO3-) is a key signaling molecule in plant metabolism and development, in addition to its role as a nutrient. It has been shown previously in Arabidopsis that ANR1, a MADS-box transcription factor, is a major component in the NO3--signaling pathway that triggers lateral root growth and that miR444, which is specific to monocots, targets four genes that are homologous to ANR1 in rice. Here, we show that miR444a plays multiple roles in the rice NO3--signaling pathway - not only in root development, but also involving nitrate accumulation and even Pi -starvation responses. miR444a overexpression resulted in reduced rice lateral root elongation, but promoted rice primary and adventitious root growth, in a nitrate-dependent manner. In addition, overexpression of miR444a improved nitrate accumulation and expression of nitrate transporter genes under high nitrate concentration conditions, but reduced the remobilization of nitrate from old leaves to young leaves thus affecting the plant's ability to adapt to nitrogen-limitating conditions. Intriguingly, we found that Pi starvation strongly induced miR444 accumulation in rice roots and that overexpression of miR444a altered Pi -starvation-induced root architecture and enhanced Pi accumulation and expression of three Pi transporter genes. We further provide evidence that miR444a is involved in the interaction between the NO3--signaling and Pi -signaling pathways in rice. Taken together, our observations demonstrated that miR444a plays multiple roles in the rice NO3--signaling pathway in nitrate-dependent root growth, nitrate accumulation and phosphate-starvation responses.

The periplasmic PDZ domain-containing protein Prc modulates full virulence, envelops stress responses, and directly interacts with dipeptidyl peptidase of Xanthomonas oryzae pv. oryzae.

PDZ domain-containing proteases, also known as HtrA family proteases, play important roles in bacterial cells by modulating disease pathogenesis and cell-envelope stress responses. These proteases have diverse functions through proteolysis- and nonproteolysis-dependent modes. Here, we report that the genome of the causative agent of rice bacterial blight, Xanthomonas oryzae pv. oryzae, encodes seven PDZ domain-containing proteins. Systematic inactivation of their encoding genes revealed that PXO_01122 and PXO_04290 (prc) are involved in virulence. prc encodes a putative HtrA family protease that localizes in the bacterial periplasm. Mutation of prc also resulted in susceptibility to multiple environmental stresses, including H2O2, sodium dodecylsulfate, and osmolarity stresses. Comparative subproteomic analyses showed that the amounts of 34 periplasmic proteins were lower in the prc mutant than in wild-type. These proteins were associated with proteolysis, biosynthesis of macromolecules, carbohydrate or energy metabolism, signal transduction, and protein translocation or folding. We provide in vivo and in vitro evidence demonstrating that Prc stabilizes and directly binds to one of these proteins, DppP, a dipeptidyl peptidase contributing to full virulence. Taken together, our results suggest that Prc contributes to bacterial virulence by acting as a periplasmic modulator of cell-envelope stress responses.

A three-component signalling system fine-tunes expression kinetics of HPPK responsible for folate synthesis by positive feedback loop during stress response of Xanthomonas campestris.

During adaptation to environments, bacteria employ two-component signal transduction systems, which contain histidine kinases and response regulators, to sense and respond to exogenous and cellular stimuli in an accurate spatio-temporal manner. Although the protein phosphorylation process between histidine kinase and response regulator has been well documented, the molecular mechanism fine-tuning phosphorylation levels of response regulators is comparatively less studied. Here we combined genetic and biochemical approaches to reveal that a hybrid histidine kinase, SreS, is involved in the SreK-SreR phosphotransfer process to control salt stress response in the bacterium Xanthomonas campestris. The N-terminal receiver domain of SreS acts as a phosphate sink by competing with the response regulator SreR to accept the phosphoryl group from the latter's cognate histidine kinase SreK. This regulatory process is critical for bacterial survival because the dephosphorylated SreR protein participates in activating one of the tandem promoters (P2) at the 5' end of the sreK-sreR-sreS-hppK operon, and then modulates a transcriptional surge of the stress-responsive gene hppK, which is required for folic acid synthesis. Therefore, our study dissects the biochemical process of a positive feedback loop in which a 'three-component' signalling system fine-tunes expression kinetics of downstream genes.