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Rice blast disease caused by infection with Magnaporthe oryzae, a hemibiotrophic fungal pathogen, significantly reduces the yield production. However, the rice defense mechanism against blast disease remains elusive. To identify the genes involved in the regulation of rice defense to blast disease, dissociation (Ds) transposon tagging mutant lines were analyzed in terms of their response to M. oryzae isolate Guy11. Among them, CBL-interacting protein kinase 31 (CIPK31) mutants were more susceptible than wild-type plants to blast. The CIPK31 transcript was found to be insensitive to Guy11 infection, and the CIPK31-GFP was localized to the cytosol and nucleus. Overexpression of CIPK31 promoted rice defense to blast. Further analysis indicated that CIPK31 interacts with Calcineurin B-like 2 (CBL2) and CBL6 at the plasma membrane, and cbl2 mutants are more susceptible to blast compared with wild-type plants, suggesting that calcium signaling might partially through the CBL2-CIPK31 signaling regulate rice defense. Yeast two-hybrid results showed that AKT1-like (AKT1L), a potential potassium (K+) channel protein, interacted with CIPK31, and the K+ level was significantly lower in the cipk31 mutants than in the wild-type control. In addition, exogenous potassium application increased rice resistance to blast, suggesting that CIPK31 might interact with AKT1L to increase K+ uptake, thereby promoting resistance to blast. Taken together, the results presented here demonstrate that CBL2-CIPK31-AKT1L is a new signaling pathway that regulates rice defense to blast disease.

Tillering is one of the most important determinants of biomass and yield in rice (Oryza sativa L.). The capacity of plants to develop tillers from primordial meristems or buds is determined not only by the genotype but also by environmental cues. Here, we characterized the function of rice grassy tiller1 (OsGT1) and its interaction with other genetic and biological factors involved in tiller bud outgrowth in rice by generating OsGT1 RNA interference (RNAi) and overexpression (OX) lines. The tiller number was increased in OsGT1-RNAi mutants but strongly suppressed in OsGT1-OX lines. Expression analysis of OsGT1 in rice phyB mutants and in genotypes carrying various genetic combinations of GT1 RNAi and phyB demonstrated that OsGT1 is not involved in phyB-mediated suppression of tiller development in rice. Expression analysis of fine culm1 (fc1), a rice tb1 homolog, and molecular assays demonstrated that FC1 enhances the expression of OsGT1 by directly binding to its promoter. Comparison of the transcriptomic profiles of fc1 and OsGT1-RNAi mutants revealed differentially expressed genes (DEGs) common to both genotypes. Finally, analysis of tillering phenotypes of OX and RNAi seedlings treated with various phytohormones implied a possible role of OsGT1 in strigolactone-mediated tiller outgrowth. Overall, this study enhances our understanding of the diverse mechanisms of tiller development in grasses.

The AMT1 family comprises major ammonium transporters in rice roots. In this study, we utilized AMT1 RNAi mutants (amt1) to explore how AMT1 affects NH4+- and NO3–-mediated morphological development and NH4+-responsive gene expression in roots. In the presence of NH4+, amt1 showed inhibition of NO3–- dependent lateral root development. The inhibitory action of NH4+ on lateral root growth was independent of the NO3– concentrations supplied to amt1 roots. The results of split root assays indicated that NH4+ exerts systemic action in inhibiting NO3–-dependent lateral root development in amt1. Further study with NAA and NOA, a potent auxin flux inhibitor, suggested that perturbation of membrane dynamics might not be the primary cause of the inhibitory action of NH4+ on NO3–-mediated lateral root growth in amt1 mutants. RNA-seq analysis of NH4+-responsive genes showed that approximately half of DEGs observed in wild-type roots were not detected in the DEGs of amt1 roots. Gene ontology enrichment analysis suggested that the expression of specific functional gene groups were affected by amt1 during the early response to NH4+. Auxin-responsive gene expression and root gravity responses were altered in amt1. This study demonstrated that AMT1 affects the interactions not only between ammonium and nitrate in lateral root growth but also between auxin and NH4+ in rice roots.

Sheath blight disease (ShB) severely affects rice production; however, the details of defense against ShB remain unclear. To understand the rice defense mechanism against ShB, an RNA sequencing analysis was performed using Rhizoctonia solani inoculated rice leaves after 48 h of inoculation. Among them, 3417 genes were upregulated and 2532 were downregulated when compared with the control group (> twofold or

Because glucose is an essential energy source for living organisms, glucose transporters (GLUTs) are present in all species worldwide. Encoded by the solute carrier family 2 gene family, the GLUT proteins generally have 12 transmembrane helices (TMHs). In total, 14 GLUT proteins have been identified in humans (hGLUTs), and they are divided into 3 classes on the basis of their transport characteristics and sequence similarities. Herein, we report the use of protein sequence similarity networks (SSNs) to visualize the sequence trends of 4101 GLUT proteins across the Metazoa. The SSNs separated the metazoan proteins into 3 new classes that were different from the traditional classification system. In the new system, 9 of the 14 hGLUTs (hGLUT1-5, 7, 9, 11, and 14) were grouped into class I, 3 (hGLUT10, 12, and 13) were grouped into class II, and 2 (hGLUT6 and 8) were grouped into class III, as also supported by the phylogenetic tree. Multiple sequence alignments further showed that the conserved residues in each class were different. Furthermore, the hGLUTs in each class showed unique evolutionary characteristics, with similar nonsynonymous-to-synonymous divergence ratios and similar regions under conservative selection pressure. Of note, GLUTs with 3, 6, 18, 24, and 36 TMHs were identified among the metazoan genomes, and 1 Chinese hamster protein with 6 TMHs showed GLUT activity. In summary, this large-scale sequence analysis provided new insights into the classification and evolution of GLUTs and further showed that gene duplication and fusion could have been important drivers during the evolution of these transporter molecules.-Jia, B., Yuan, D. P., Lan, W. J., Xuan, Y. H., Jeon, C. O. New insight into the classification and evolution of glucose transporters in the Metazoa.

Background Sheath blight disease (ShB) is a destructive disease affecting rice production. Previously, we have reported that Loose Plant Architecture 1 (LPA1) promotes resistance to ShB. However, the mechanisms by which LPA1 confers resistance against this disease have not been extensively investigated. Notably, interactors that regulate LPA-1 activity remain elusive. Findings In this study, we identified the interaction of kinesin-like protein (KLP) with LPA1 in the nucleus of rice cells by yeast two-hybrid, bimolecular fluorescent complimentary (BiFC), and co-immunoprecipitation (co-IP) assays. To investigate the role of KLP in promoting resistance to ShB, wild-type, klp mutant, and KLP overexpressor (KLP OX) rice plants were inoculated with Rhizoctonia solani AG1-IA. The results indicated that, compared with the wild-type control, klp mutants were more susceptible while KLP OX plants were less susceptible to ShB. Since LPA1 transcriptionally activates PIN-FORMED 1a (PIN1a), we examined the expression of 8 related PIN genes. The results showed that only the expression of PIN1a and PIN3b coincided with KLP expression levels. In addition, a chromatin immunoprecipitation (ChIP) assay showed that KLP bound directly to the promoter region of PIN1a but not of PIN3b. Transient expression assays confirmed that LPA1 and KLP transcriptionally activate PIN1a, and that coexpression of KLP and LPA1 had an additive effect on the activation of PIN1a, suggesting that KLP enhances LPA1 transcriptional activation activity. Conclusions Taken together, our results show that KLP is a novel LPA1 interactor that promotes resistance of rice to ShB.

Rice (Oryza sativa) production is damaged to a great extent by sheath blight disease (ShB). However, the defense mechanism in rice against this disease is largely unknown. Previous transcriptome analysis identified a significantly induced eukaryotic protein phosphatase 2A catalytic subunit 1 (PP2A-1) after the inoculation of Rhizoctonia solani. Five genes encoding PP2A exist in rice genome, and these five genes are ubiquitously expressed in different tissues and stages. Inoculation of R. solani showed that the genome edited pp2a-1 mutants using the CRISPR/Cas9 were more susceptible to ShB than the wild-type control, but other PP2A gene mutants exhibited similar response to ShB compared to wild-type plants. In parallel, PP2A-1 expression level was higher in the activation tagging line, and PP2A-1 overexpression inhibited plant height and promoted the resistance to ShB. PP2A-1-GFP was localized in the cytoplasm and nucleus. In addition, R. solani-dependent induction kinetics of pathogen-related genes PBZ1 and PR1b was lower in pp2a-1 mutants but higher in PP2A-1 activation line compared to those in the wild-type. In conclusion, our analysis shows that PP2A-1 is a member of protein phosphatase, which regulates rice resistance to ShB. This result broadens the understanding of the defense mechanism against ShB and provides a potential target for rice breeding for disease resistance.

Plant architecture and disease resistance are the key factors that control the production of yield. However, the mechanism behind these factors is largely unknown. In this study, we identified that indeterminate domain 3 (IDD3) was obviously induced by inoculation of Rhizoctonia solani AG1-IA. Plants that overexpressed IDD3 (IDD3 OX) were more susceptible, while idd3 mutants showed a similar response to sheath blight disease compared with wild-type plants. Interestingly, IDD3 OX plants developed a wider tiller angle and exhibited altered shoot gravitropism, while idd3 knock-out mutants showed no visible morphological differences compared with the wild-type plants. IDD3 is ubiquitously expressed in different tissues and stages, and the IDD3 transcript was induced by exogenously applied auxin. Expression of the PIN-FORMED (PIN) and Aux/IAA genes was altered in IDD3 OX compared with wild-type plants. Furthermore, IDD3 OX plants are sensitive to auxin and the polar auxin transporter inhibitor N-1-naphthylphalamic acid (NPA). Further yeast-one hybrid, chromatin immunoprecipitation (ChIP) and transient assays revealed that IDD3 directly represses PIN1b via promoter binding. Inoculation with R. solani indicated that PIN1b RNAi plants are more susceptible to sheath blight disease (ShB) compared with the wild-type. Taken together, our analyses suggest that IDD3 controls plant architecture and the resistance of rice to ShB via the regulation of PIN auxin transporter genes.

Root-knot nematodes (RKNs) severely affect plants growth and productivity, and several commercial biocontrol bacteria can improve plants resistance to RKNs. Pseudomonas putida Sneb821 isolate was found to induce tomatoes resistance against Meloidogyne incognita. However, the molecular functions behind induced resistance remains unclear. Long non-coding RNA (lncRNA) is considered to be a new component that regulates the molecular functions of plant immunity. We found lncRNA was involved in Sneb821-induced tomato resistance to M. incognita. Compared with tomato inoculated with M. incognita, high-throughput sequencing showed that 43 lncRNAs were upregulated, while 35 lncRNAs were downregulated in tomatoes previously inoculated with Sneb821. A regulation network of lncRNAs was constructed, and the results indicated that 12 lncRNAs were found to act as sponges of their corresponding miRNAs. By using qRT-PCR and the overexpression vector pBI121, we found the expression of lncRNA44664 correlated with miR396/GRFs (growth-regulating factors) and lncRNA48734 was correlated with miR156/SPL (squamosal promoter-binding protein-like) transcription factors. These observations provided a novel molecular model in biocontrol bacteria-induced tomato resistance to M. incognita.

Meloidogyne incognita and Meloidogyne graminicola are root-knot nematodes (RKNs) infecting rice (Oryza sativa L.) roots and severely decreasing yield, whose mechanisms of action remain unclear. We investigated RKN invasion and development in rice roots through RNA-seq transcriptome analysis. The results showed that 952 and 647 genes were differently expressed after 6 (invasion stage) and 18 (development stage) days post inoculation, respectively. Gene annotation showed that the differentially expressed genes were classified into diverse metabolic and stress response categories. Furthermore, phytohormone, transcription factor, redox signaling, and defense response pathways were enriched upon RKN infection. RNA-seq validation using qRT-PCR confirmed that CBL-interacting protein kinase (CIPK) genes (CIPK5, 8, 9, 11, 14, 23, 24, and 31) as well as brassinosteroid (BR)-related genes (OsBAK1, OsBRI1, D2, and D11) were altered by RKN infection. Analysis of the CIPK9 mutant and overexpressor indicated that the RKN populations were smaller in cipk9 and larger in CIPK9 OX, while more galls were produced in CIPK9 OX plant roots than the in wild-type roots. Significantly fewer numbers of second-stage infective juveniles (J2s) were observed in the plants expressing the BR biosynthesis gene D2 mutant and the BR receptor BRI1 activation-tagged mutant (bri1-D), and fewer galls were observed in bri1-D roots than in wild-type roots. The roots of plants expressing the regulator of ethylene signaling ERS1 (ethylene response sensor 1) mutant contained higher numbers of J2s and developed more galls compared with wild-type roots, suggesting that these signals function in RKN invasion or development. Our findings broaden our understanding of rice responses to RKN invasion and provide useful information for further research on RKN defense mechanisms.

Rhizoctonia solani causes sheath blight disease in rice; however, the defense mechanism of rice plants against R. solani remains elusive. To analyze the roles of brassinosteroid (BR) and ethylene signaling on rice defense to R. solani, wild-type (WT) rice and several mutants and overexpressing (OX) lines were inoculated with R. solani. Mutants d61-1 and d2 were less susceptible to sheath blight disease, bri1-D was more susceptible, and ravl1 and d61-1/EIL1 Ri5 were similarly susceptible compared with WT. The double mutant ravl1/d61-1 was phenotypically similar to the ravl1 mutant. Transcriptome analysis, chromatin immunoprecipitation assay, electrophoretic mobility shift assay, and transient assays indicted that RAVL1 might directly activate Ethylene insensitive 3-like 1 (EIL1), a master regulator of ethylene signaling. Mutants ers1 and d61-1/RAVL1 OX were resistant to sheath blight disease, whereas EIL1 RNAi mutants and RAVL1 OX were more susceptible than WT. BRI1 and D2 expression in EIL1 Ri5/RAVL1 OX and EIL1 expression in d61-1/RAVL1 OX indicated that RAVL1 activates BRI1/D2 and EIL1, respectively, independent of BR and ethylene signaling. Our analyses provide information on how BR and ethylene signaling regulate sheath blight disease and on the regulatory function of RAVL1 in rice sheath blight disease.

Pathogen–host interaction is a complicated process; pathogens mainly infect host plants to acquire nutrients, especially sugars. Rhizoctonia solani, the causative agent of sheath blight disease, is a major pathogen of rice. However, it is not known how this pathogen obtains sugar from rice plants. In this study, we found that the rice sugar transporter OsSWEET11 is involved in the pathogenesis of sheath blight disease. Quantitative real‐time polymerase chain reaction (qRT‐PCR) and β‐d‐glucuronidase expression analyses showed that R. solani infection significantly enhanced OsSWEET11 expression in leaves amongst the clade III SWEET members. The analyses of transgenic plants revealed that Ossweet11 mutants were less susceptible, whereas plants overexpressing OsSWEET11 were more susceptible, to sheath blight compared with wild‐type controls, but the yield of OsSWEET11 mutants and overexpressors was reduced. SWEETs become active on oligomerization. Split‐ubiquitin yeast two‐hybrid, bimolecular fluorescence complementation and co‐immunoprecipitation assays showed that mutated OsSWEET11 interacted with normal OsSWEET11. In addition, expression of conserved residue mutated AtSWEET1 inhibited normal AtSWEET1 activity. To analyse whether inhibition of OsSWEET11 function in mesophyll cells is related to defence against this disease, mutated OsSWEET11 was expressed under the control of the Rubisco promoter, which is specific for green tissues. The resistance of transgenic plants to sheath blight disease, but not other disease, was improved, whereas yield production was not obviously affected. Overall, these results suggest that R. solani might acquire sugar from rice leaves by the activation of OsSWEET11 expression. The plants can be protected from infection by manipulation of the expression of OsSWEET11 without affecting the crop yield.

The root-knot nematode (RKN) severely affects plant growth and productivity. Thus, protection of plants from RKNs has become an important task, especially for increasing crop yields. The objective of this study was to screen plant growth-promoting rhizobacteria (PGPR) for the ability to control RKNs. In this study, 860 strains of bacteria were collected from rhizosphere soil and five bacteria isolates were identified with high efficacy against RKNs: Bacillus cereus (Sneb 560), B. subtilis (Sneb 815), Pseudomonas putida (Sneb 821), P. fluorescens (Sneb 825) and Serratia proteamaculans (Sneb 851). The five bacteria isolates had high larvicidal and ovicidal activity in vitro. In a novel result, Sneb 851 showed especially high potential as a biocontrol agent against Meloidogyne incognita, causing 99.17% juvenile mortality and 61.11% egg mortality. In the pot experiment, tomato seeds treated with Sneb 825 displayed significantly higher levels of growth in root and shoot compared to control plants. Meanwhile, treatment with Sneb 815, Sneb 821 and Sneb 825 exhibited higher efficacies in reducing the number of galls and juveniles in the soil. In the field experiment, application of the five bacteria isolates increased the plant biomass and showed high biocontrol efficacy against M. incognita. The lowest gall index was observed in the treatment with Sneb 815 and Sneb 825 both in 2014 and 2015. Taken together, the five PGPR isolates can be regarded as potential biocontrol agents against RKNs in the future.

NH4+ is an important nitrogen resource for rice plants in paddy soil. Therefore, it is likely that NH4+-triggered plant growth interacts with phytohormone-mediated developmental mechanisms. Our previous transcriptomic analysis revealed that many genes involved in auxin signaling and efflux are sensitive to NH4+. In the current study, we found that NH4+ treatment causes a delayed gravity response in rice roots. To further elucidate the interlocking relationship between NH4+ and auxin signaling during root development, we utilized mutants and overexpressors of a key NH4+ signaling transcription factor INDETERMINATE DOMAIN 10 (IDD10), encoding a transcription factor that regulates the expression of NH4+ uptake and N-assimilation genes. We obtained several lines of evidence that auxin affects NH4+-mediated gene expression and root development in rice plants via IDD10. First, the gravity response was delayed in idd10 roots and accelerated in IDD10 overexpressor (IDD10 OX) roots in the absence and (especially) presence of NH4+. Second, idd10 plants showed strong root coiling only in the presence of NH4+. However, treatment of 1-N-naphthylphthalamic acid (NPA), a polar auxin transport inhibitor suppressed the NH4+-specific root phenotype of idd10. Third, the expression of NH4+-responsive auxin-related genes was affected in idd10 and IDD10 overexpressors. Finally, IDD10 expression was induced by IAA and suppressed by NPA. These findings suggest that the gene expression patterns and phenotypes triggered by NH4+ are influenced by the actions of auxin during root development, pointing to a regulatory circuit between NH4+ and auxin signaling that functions in root development in rice.

Glycine max (soybean) is an extremely important crop, representing a major source of oil and protein for human beings. Heterodera glycines (soybean cyst nematode, SCN) infection severely reduces soybean production; therefore, protecting soybean from SCN has become an issue for breeders. Black soybean has exhibited a different grade of resistance to SCN. However, the underlying mechanism of Huipizhi Heidou resistance against SCN remains elusive. The Huipizhi Heidou (ZDD2315) and race 3 of Heterodera glycines were chosen to study the mechanism of resistance via examination of transcriptomic changes. After 5, 10, and 15days of SCN infection, whole roots were sampled for RNA extraction, and uninfected samples were simultaneously collected as a control. 740, 1413, and 4925 genes were isolated by padj (p-value adjusted)

NH 4 + is important for the growth and the yield production of paddy-soil grown rice. However, the mechanism of NH 4 + signaling is largely unknown. Previously, we performed a microarray analysis to examine genomic responses and signaling pathways in rice roots exposed to NH 4 + , and interestingly found that transcription of a large number of genes including kinase, phosphatase, and transcription factors were sensitive to NH 4 + . In this study, we further analyzed transcription factors respond to NH 4 + , and identified an NH 4 + -repressed bHLH transcription factor involved in NH 4 + acquisition process. bHLH is a nuclear protein and has transcription-activation activity in yeast and plants. RNAi-mediated suppression of bHLH and bHLH overexpression ( bHLH OX ) resulted in the accumulation of lower and higher NH 4 + contents in transgenic rice roots, respectively. NH 4 + -mediated induction of AMT1 ; 1 and AMT1 ; 2 is lower in bHLH RNAi , but higher in bHLH OX as compared to in wild-type plant roots. However, bHLH does not directly activate AMT1 ; 1 and AMT1 ; 2 transcriptions. IDD10 expression is not altered in bHLH RNAi plant roots, and bHLH maintained similar expression level in idd10 mutant roots compared to in wild-type plant roots. Furthermore, NH 4 + contents in idd10 ; bHLH RNAi double mutant roots were much lower than in each single mutant and wild-type plant roots, indicating that IDD10 and bHLH independently regulates NH 4 + uptake. Taken together, these results provide a new regulatory path by which bHLH regulates NH 4 + uptake in rice.

The Fusarium graminearum species complex (FGSC) is an important group of pathogens distributed in maize-producing areas worldwide. This study investigated the genetic diversity and pathogenicity of 40 FGSC isolates obtained from stalk rot and ear rot samples collected from 42 locations in northeastern China during 2013 and 2014. A phylogenetic tree of translation elongation factor (EF-la) sequences designated the 40 isolates as F. graminearum sensu stricto (67.5%) and F. boothii (32.5%). By using inter-simple sequence repeat analysis (ISSR), it was shown that the isolates were divided into two clades, which corresponded to the species identity of the isolates. However, the isolates from the two different diseases could not be distinguished in pathogenicity. The disease severity index of seedlings inoculated with stalk isolates was slightly higher than that of seedlings inoculated with isolates from infected ears, whereas the pathogenicity of the stalk and ear isolates were identical.

Rice (Oryza sativa) is the most important consumed staple food for a large and diverse population worldwide. Since databases of genomic sequences became available, functional genomics and genetic manipulations have been widely practiced in rice research communities. Insertional mutants are the most common genetic materials utilized to analyze gene function. To mutagenize rice genomes, we exploited the transpositional activity of an Activator/Dissociation (Ac/Ds) system in rice. To mobilize Ds in rice genomes, a maize Ac cDNA was expressed under the CaMV35S promoter, and a gene trap Ds was utilized to detect expression of host genes via the reporter gene GUS. Conventional transposon-mediated gene-tagging systems rely on genetic crossing and selection markers. Furthermore, the activities of transposases have to be monitored. By taking advantage of the fact that Ds becomes highly active during tissue culture, a plant regeneration system employing tissue culture was employed to generate a large Ds transposant population in rice. This system overcomes the requirement for markers and the monitoring of Ac activity. In the regenerated populations, more than 70% of the plant lines contained independent Ds insertions and 12% expressed GUS at seedling stages. This protocol describes the method for producing a Ds-mediated insertional population via tissue culture regeneration systems. © 2016 by John Wiley & Sons, Inc.

Cardiac-specific metallothionein (MT) overexpression extends lifespan, but the mechanism underlying the effect of MT protection against age-associated cardiovascular diseases (CVD) remains elusive. To elucidate this, male wild-type and two lines of MT-transgenic (MT-TG) mice, MM and MT-1 (cardiac-specific overexpressing MT about 10- and 80-fold, respectively) at three representative ages (2-3, 9-10, and 18-20 months), were utilized. A stable human MT2A overexpressing cardiomyocytes (H9c2MT7) was also introduced.Histomorphology and echocardiographic analysis revealed that age-associated cardiac hypertrophy, remodeling, and dysfunction were ameliorated in MT-TG mice. Also, aging-accompanied NF-κB activation, characterized by increased nuclear p65 translocation, elevated DNA-binding activity, and upregulation of inflammatory cytokines, was largely attenuated by MT overexpression. Treatment of H9c2 cardiomyocytes with tumor necrosis factor-α (TNF-α), which mimicked an inflammatory environment, significantly increased NF-κB activity, and some age-related phenotypes appeared. The NF-κB activation was further proved to be pivotal for both age-associated and TNF-α-induced nitrative damage to cardiac 2-oxoglutarate dehydrogenase (2-OGD) by virtue of NF-κB p65 gene silencing. MT inhibited NF-κB activation and associated nitrative damage to cardiac 2-OGD in both old MT-TG hearts and TNF-α-treated H9c2MT7 cardiomyocytes; these protective effects were abolished in H9c2MT7 cardiomyocytes by MT-specific gene silencing. Innovation and Conclusion: Together, these findings indicate that the protective effects of MT against age-associated CVD can be attributed mainly to its role in NF-κB inhibition and resultant alleviation of nitrative damage to 2-OGD. Antioxid. Redox Signal. 25, 936-952.

Several studies have been performed to elucidate the role of brassinosteroids (BRs) in plant growth and development. However, information on the role of BR signaling in nutrient uptake is limited. This study explores the relationship between BRs and ammonium transporter 1 (AMT1) expression in Arabidopsis roots. We found that BR treatment reduced the expression of AMT1 genes and that a BR receptor BRI1 mutant bri1-5 reversed its BR-repressed expression. Furthermore, the BR signaling transcription factor, BES1, regulates AMT1 expression in roots. NH4 +-mediated repression of AMT1;1, AMT1;2, and AMT1;3 was suppressed in a gain-of-function BES1 mutant (bes1-D). This mutant was more sensitive to methyl-ammonium and contained a higher ammonium content compared to wild-type plants. However, BES1 failed to bind E-box elements present in the promoter region of the AMT1 genes. Furthermore, NH4 +-mediated glutamine synthetase (GS) and glutamine oxoglutarate aminotransferase (GOGAT) gene expressions were partially inhibited, and GS activity was slightly lower in the bes1-D mutant relative to that observed in wild-type En2 roots. NH4 +-mediated AMT1 suppressions are known to be caused by N-metabolites rather than NH4 + itself, and glutamine application inhibited AMT1 expression in both En2 and bes1-D indicating that BES1 activation inhibited NH4 +-mediated GS/GOGAT induction, which might in turn inhibit AMT1 repression. In conclusion, the present study demonstrates that BR regulated nitrogen uptake and assimilation via the BR signaling pathway.

In plant immune responses, reactive oxygen species (ROS) act as signaling molecules that activate defense pathways against pathogens, especially following resistance (R) gene-mediated pathogen recognition. Glutathione (GSH), an antioxidant and redox regulator, participates in the removal of hydrogen peroxide (H2O2). However, the mechanism of GSH-mediated H2O2 generation in soybeans (Glycine max (L.) Merr.) that are resistant to the soybean cyst nematode (SCN, Heterodera glycines Ichinohe) remains unclear. To elucidate this underlying relationship, the feeding of race 3 of H. glycines with resistant cultivars, Peking and PI88788, was compared with that on a susceptible soybean cultivar, Williams 82. After 5, 10, and 15 days of SCN infection, we quantified &gamma, glutamylcysteine (&gamma, EC) and (homo)glutathione ((h)GSH), and a gene expression analysis showed that GSH metabolism in resistant cultivars differed from that in susceptible soybean roots. ROS accumulation was examined both in resistant and susceptible roots upon SCN infection. The time of intense ROS generation was related to the differences of resistance mechanisms in Peking and PI88788. ROS accumulation that was caused by the (h)GSH depletion-arrested nematode development in susceptible Williams 82. These results suggest that (h)GSH metabolism in resistant soybeans plays a key role in the regulation of ROS-generated signals, leading to resistance against nematodes.

Background and Aims INDETERMINATE DOMAIN 10 (IDD10) is a key transcription factor gene that activates the expression of a large number of NH4+-responsive genes including AMMONIUM TRANSPORTER 1;2 (AMT1;2). Primary root growth of rice (Oryza sativa) idd10 mutants is hypersensitive to NH4+. The involvement of CALCINEURIN B-LIKE INTERACTING PROTEIN KINASE (CIPK) genes in the action of IDD10 on NH4+-mediated root growth was investigated. Methods Quantitative reverse transcription–PCR was used to analyse NH4+- and IDD10-dependent expression of CIPK genes. IDD10-regulated CIPK target genes were identified using electrophoretic mobility shift assays, chromatin immunoprecipitation and transient transcription assays. Root growth rate, ammonium content and 15N uptake of cipk mutants were measured to determine their sensitivity to NH4+ and to compare these phenotypes with those of idd10. The genetic relationship between CIPK9 OX and idd10 was investigated by crosses between the CIPK9 and IDD10 lines. Key Results AMT1;2 was overexpressed in idd10 to determine whether NH4+-hypersensitive root growth of idd10 resulted from limitations in NH4+ uptake or from low cellular levels of NH4+. High NH4+ levels in idd10/AMT1;2 OX did not rescue the root growth defect. Next, the involvement of CIPK genes in NH4+-dependent root growth and interactions between IDD10 and CIPK genes was investigated. Molecular analysis revealed that IDD10 directly activated transcription of CIPK9 and CIPK14. Expression of CIPK8, 9, 14/15 and 23 was sensitive to exogenous NH4+. Further studies revealed that cipk9 and idd10 had almost identical NH4+-sensitive root phenotypes, including low efficiency of 15NH4+ uptake. Analysis of plants containing both idd10 and CIPK9 OX showed that CIPK9 OX could rescue the NH4+-dependent root growth defects of idd10. Conclusions CIPK9 was involved in NH4+-dependent root growth and appeared to act downstream of IDD10. This information will be useful in future explorations of NH4+ signalling in plants.

Soybean is an important economic crop that is often adversely affected by infection in the field with the soybean cyst nematode Heterodera glycines. Biological control is an eco-friendly method used to protect the crop against disease. The bacterium Klebsiella pneumoniae has been reported to protect rice from sheath blight and seedling blight, but its role in the control of nematode is unclear. In this study, the effect of K. pneumoniae SnebYK on the control of H. glycines was assessed. Potting experiment results showed that coating soybean seeds with K. pneumoniae SnebYK not only reduced the infection rate of H. glycines but also decreased the proportion of adult female nematodes. Field experiment results showed that K. pneumoniae SnebYK reduced both the number of H. glycines in soybean roots and the number of adult females. However, K. pneumoniae SnebYK caused low juvenile mortality in an in vitro assay. To further analyze the role of K. pneumoniae SnebYK in the inhibition of H. glycines infection, split root experiments were conducted. The results indicated that K. pneumoniae SnebYK controls H. glycines via induced systemic resistance, which reduces H. glycines penetration. Klebsiella pneumoniae SnebYK treatment also significantly increased the proportion of second-stage juveniles and decreased the proportions of third- and fourth-stage juveniles in the H. glycines population. Moreover, 48 h after inoculation with H. glycines, the expression levels of PR1, PR2, PR5, and PDF1.2 were significantly higher in soybeans pretreated with K. pneumoniae SnebYK than in control soybeans. Interestingly, besides providing protection against nematodes, K. pneumoniae SnebYK fixed nitrogen, produced ammonia, solubilized phosphate, and produced siderophores, leading to well-developed root system and an increase in soybean seedling fresh weight. These results demonstrate for the first time that K. pneumoniae SnebYK not only promotes soybean growth but also inhibits the invasion and development of H. glycines by inducing systemic resistance.

Sugars will eventually be exported transporters (SWEETs) and SemiSWEETs represent a family of sugar transporters in eukaryotes and prokaryotes, respectively. SWEETs contain seven transmembrane helices (TMHs), while SemiSWEETs contain three. The functions of SemiSWEETs are less studied. In this perspective article, we analyzed the diversity and conservation of SemiSWEETs and further proposed the possible functions. 1,922 SemiSWEET homologs were retrieved from the UniProt database, which is not proportional to the sequenced prokaryotic genomes. However, these proteins are very diverse in sequences and can be classified into 19 clusters when >50% sequence identity is required. Moreover, a gene context analysis indicated that several SemiSWEETs are located in the operons that are related to diverse carbohydrate metabolism. Several proteins with seven TMHs can be found in bacteria, and sequence alignment suggested that these proteins in bacteria may be formed by the duplication and fusion. Multiple sequence alignments showed that the amino acids for sugar translocation are still conserved and coevolved, although the sequences show diversity. Among them, the functions of a few amino acids are still not clear. These findings highlight the challenges that exist in SemiSWEETs and provide future researchers the foundation to explore these uncharted areas.

Plasmodiophora brassicae is a soil borne pathogen and the causal agent of clubroot, a devastating disease of Brassica crops. The pathogen lives inside roots, and hijacks nutrients from the host plants. It is suggested that clubroot galls created an additional nutrient sink in infected roots. However, the molecular mechanism underlying P. brassicae infection and sugar transport is unclear. Here, we analyzed sugar contents in leaves and roots before and after P. brassicae infection using a pair of Chinese cabbage near-isogenic lines (NILs), carrying either a clubroot resistant (CR) or susceptible (CS) allele at the CRb locus. P. brassicae infection caused significant increase of glucose and fructose contents in the root of CS-NIL compared to CR-NIL, suggesting that sugar translocation and P. brassicae growth are closely related. Among 32 B. rapa SWEET homologs, several BrSWEETs belonging to Clade I and III were significantly up-regulated, especially in CS-NIL upon P. brassicae infection. Moreover, Arabidopsis sweet11 mutant exhibited slower gall formation compared to the wild-type plants. Our studies suggest that P. brassicae infection probably triggers active sugar translocation between the sugar producing tissues and the clubbed tissues, and the SWEET family genes are involved in this process.

Although pathogens such as nematodes are known to hijack nutrients from host plants, the mechanisms whereby nematodes obtain sugars from plants remain largely unknown. To determine the effects of nematode infection on host plant sugar allocation, soluble sugar (fructose, glucose, sucrose) content was investigated using high-performance liquid chromatography with refractive index detection and was found to increase significantly in tomato (Solanum lycopersicum, Sl) leaves and roots during early infection by root-knot nematodes (RKNs). To further analyze whether sugar transporters played a role in this process, the expression levels of sucrose transporter (SUT/SUC), Sugars Will Eventually be Exported Transporter (SWEET), tonoplast monosaccharide transporter (TMT), and vacuolar glucose transporter (VGT) gene family members were examined by qRT-PCR analysis after RKN infection. The results showed that three SlSUTs, 17 SlSWEETs, three SlTMTs, and SlVGT1 were upregulated in the leaves, whereas three SlSUTs, 17 SlSWEETs, two SlTMTs, and SlVGT1 were induced in the roots. To determine the function of the sugar transporters in the RKN infection process, we examined post-infection responses in the Atsuc2 mutant and pAtSUC2-GUS lines. β-glucuronidase expression was strongly induced at the infection sites, and RKN development was significantly arrested in the Atsuc2 mutant. Taken together, our analyses provide useful information for understanding the sugar transporter responses during early infection by RKNs in tomato.

Sugars Will Eventually be Exported Transporter (SWEET) and SemiSWEET are recently characterized families of sugar transporters in eukaryotes and prokaryotes, respectively. SemiSWEETs contain 3 transmembrane helices (TMHs), while SWEETs contain 7. Here, we performed sequence-based comprehensive analyses for SWEETs and SemiSWEETs across the biosphere. In total, 3,249 proteins were identified and ≈60% proteins were found in green plants and Oomycota, which include a number of important plant pathogens. Protein sequence similarity networks indicate that proteins from different organisms are significantly clustered. Of note, SemiSWEETs with 3 or 4 TMHs that may fuse to SWEET were identified in plant genomes. 7-TMH SWEETs were found in bacteria, implying that SemiSWEET can be fused directly in prokaryote. 15-TMH extraSWEET and 25-TMH superSWEET were also observed in wild rice and oomycetes, respectively. The transporters can be classified into 4, 2, 2, and 2 clades in plants, Metazoa, unicellular eukaryotes, and prokaryotes, respectively. The consensus and coevolution of amino acids in SWEETs were identified by multiple sequence alignments. The functions of the highly conserved residues were analyzed by molecular dynamics analysis. The 19 most highly conserved residues in the SWEETs were further confirmed by point mutagenesis using SWEET1 from Arabidopsis thaliana. The results proved that the conserved residues located in the extrafacial gate (Y57, G58, Y83, G131, and P191), the substrate binding pocket (N73, N192, and W176), and the intrafacial gate (P43, F87, P145, M161, P162, and Q202) play important roles for substrate recognition and transport processes. Taken together, our analyses provide a foundation for understanding the diversity, classification, and evolution of SWEETs and SemiSWEETs using large-scale sequence analysis and further show that that gene duplication and gene fusion are important factors driving the evolution of SWEETs.

Wheat stem rust, caused byPuccinia granimisf. sp.tritici, severely affects wheat production, but it has been effectively controlled in China since the 1970s. However, the appearance and spread of wheat stem rust races Ug99 (TTKSK, virulence toSr31), TKTTF (virulence toSrTmp) and TTTTF (virulence to the cultivars carryingSr9eandSr13) have received attention. It is important to clarify the effectiveness of resistance genes in a timely manner, especially for the purpose of using new resistance genes in wheat cultivars for durable-resistance. However, little is known about the stem rust resistance genes present in widely used wheat cultivars from Gansu. This study aimed to determine the resistance level at the seedling stage of the main wheat cultivars in Gansu Province. A secondary objective was to assess the prevalence ofSr2,Sr24,Sr25,Sr26,Sr31, andSr38using molecular markers. The results of the present study indicated that 38 (50.7%) wheat varieties displayed resistance to all the tested races ofPuccinia graminisf. sp.tritici.The molecular marker analysis showed that 13 out of 75 major wheat cultivars likely carriedSr2; 25 wheat cultivars likely carriedSr31; and nine wheat cultivars likely carriedSr38. No cultivar was found to haveSr25andSr26, as expected. Surprisingly, no wheat cultivars carriedSr24. The wheat lines with known stem rust resistance genes could be used as donor parent for further breeding programs.

The SWEET (sugars will eventually be exported transporter) family is a newly characterized group of sugar transporters. In plants, the key roles of SWEETs in phloem transport, nectar secretion, pollen nutrition, stress tolerance, and plant-pathogen interactions have been identified. SWEET family genes have been characterized in many plant species, but a comprehensive analysis of SWEET members has not yet been performed in wheat. Here, 59 wheat SWEETs (hereafter TaSWEETs) were identified through homology searches. Analyses of phylogenetic relationships, numbers of transmembrane helices (TMHs), gene structures, and motifs showed that TaSWEETs carrying 3-7 TMHs could be classified into four clades with 10 different types of motifs. Examination of the expression patterns of 18 SWEET genes revealed that a few are tissue-specific while most are ubiquitously expressed. In addition, the stem rust-mediated expression patterns of SWEET genes were monitored using a stem rust-susceptible cultivar, 'Little Club' (LC). The resulting data showed that the expression of five out of the 18 SWEETs tested was induced following inoculation. In conclusion, we provide the first comprehensive analysis of the wheat SWEET gene family. Information regarding the phylogenetic relationships, gene structures, and expression profiles of SWEET genes in different tissues and following stem rust disease inoculation will be useful in identifying the potential roles of SWEETs in specific developmental and pathogenic processes.

This study investigated the role of the sugar transporter OsSWEET11 during the early stage of rice caryopsis development using β-glucoronidase (GUS) to represent its expression, together with clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9)-mediated knockout, cross-fertilization and RNA sequencing (RNA-seq) analyses. The results showed that OsSWEET11 was expressed strongly in developing caryopsis, particularly in the ovular vascular trace, nucellar epidermis and cross cells. The knockout of OsSWEET11 significantly decreased the sucrose concentration in the mutant embryo sacs and led to defective grain filling compared with that of the wild-type (WT) plant. Moreover, the expression of 2,549 genes in the mutant caryopsis was affected. The grain weight and seed setting percentage were also decreased in the mutants. The cross-fertilization of the mutant and WT rice revealed that the mutated maternal donor induced defective grain filling. These results strongly suggested that OsSWEET11 played an important role in sucrose release from maternal tissue to the maternal-filial interface during the early stage of caryopsis development. It might also induce sucrose release from the ovular vascular trace and cross cells of developing caryopsis. These findings bridge the gap in the understanding of post-phloem sugar transport during the early stage of rice caryopsis development.

Skin wound healing is a complex process requiring the coordinated behavior of many cell types, especially in the proliferation and migration of fibroblasts. Basic fibroblast growth factor (bFGF) is a member of the FGF family that promotes fibroblast migration, but the underlying molecular mechanism remains elusive. The present RNA sequencing study showed that the expression levels of several canonical Wnt pathway genes, including Wnt2b, Wnt3, Wnt11, T-cell factor 7 (TCF7), and Frizzled 8 (FZD8) were modified by bFGF stimulation in fibroblasts. Enzyme-linked immunosorbent assay (ELISA) analysis also showed that Wnt pathway was activated under bFGF treatment. Furthermore, treatment of fibroblasts with lithium chloride or IWR-1, an inducer and inhibitor of the Wnt signaling pathway, respectively, promoted and inhibited cell migration. Also, levels of cytosolic glycogen synthase kinase 3 beta phosphorylated at serine9 (pGSK3β Ser9) and nuclear β-catenin were increased upon exposure to bFGF. Molecular and biochemical assays indicated that phosphoinositide 3-kinase (PI3K) signaling activated the GSK3β/β-catenin/Wnt signaling pathway via activation of c-Jun N-terminal kinase (JNK), suggesting that PI3K and JNK act at the upstream of β-catenin. In contrast, knock-down of β-catenin delayed fibroblast cell migration even under bFGF stimulation. RNA sequencing analysis of β-catenin knock-down fibroblasts demonstrated that β-catenin positively regulated the transcription of bFGF and FGF21. Moreover, FGF21 treatment activated AKT and JNK, and accelerated fibroblast migration to a similar extent as bFGF does. In addition, ELISA analysis demonstrated that both of bFGF and FGF21 were auto secretion factor and be regulated by Wnt pathway stimulators. Taken together, our analyses define a feedback regulatory loop between bFGF (FGF21) and Wnt signaling acting through β-catenin in skin fibroblasts.

Soybean cyst nematode (SCN) causes heavy losses to soybean yield. In order to investigate the roles of soybean miRNAs during the early stages of infection (1 and 5 dpi), 24 small RNA libraries were constructed from SCN resistant cultivar Huipizhi (HPZ) and the susceptible Williams 82 (W82) cultivar for high-throughput sequencing. By sequencing the small RNA libraries, a total of 634 known miRNAs were identified, and 252 novel miRNAs were predicted. Altogether, 14 known miRNAs belonging to 13 families, and 26 novel miRNAs were differentially expressed and may respond to SCN infection in HPZ and W82. Similar expression results were also confirmed by qRT-PCR. Further analysis of the biological processes that these potential target genes of differentially expressed miRNAs regulate found that they may be strongly related to plant&ndash, pathogen interactions. Overall, soybean miRNAs experience profound changes in early stages of SCN infection in both HPZ and W82. The findings of this study can provide insight into miRNAome changes in both HPZ and W82 at the early stages of infection, and may provide a stepping stone for future SCN management.

Sheath blight disease (ShB) in rice is caused by Rhizoctonia solani. However, there have been no major genes identified from wild rice relatives and cultivated rice that encode resistance to R. solani. In this study, 300 Ds tagging mutants were generated via tissue culture regeneration methods from a Ds (dissociation) insertion line, and R. solani AG1-IA was inoculated to isolate the resistant mutants. The starter line that was homozygous (Ds-s) with a single Ds insertion in chromosome 1 exhibited a similar response to ShB as the non-transgenic plants. A Southern blot analysis showed that the Ds element was actively transposed in 300 R1 lines generated from Ds-s, and R2 plants were generated by selfing using 300 R1 lines with Ds insertions. Out of 300 R2 lines, two lines displayed susceptibility, and two lines exhibited strong resistance to ShB. In addition, 23 mutant lines displayed resistant symptoms, and the remaining 273 lines showed no differences compared to the ShB-susceptible wild-type control plants. Further examination revealed that the Ds gene was inserted at the second intron of either the PHYTOCHROME B (PHYB) or LAZY1 gene in two strong resistant mutants. We showed that the ethylene signaling genes (EIL1, EIL2, EIN2, and ERF63), jasmonic acid synthesis gene (LOX2), and salicylic acid signaling gene PBZ1 were up-regulated in the phyB mutants compared with the wild-type control that is susceptible to ShB. Further genetic study using EIL1 RNAi and EIL1 overexpression plants revealed that EIL1 positively regulates the resistance of rice to ShB. Overall, our analyses provided a basis for the identification of ShB resistance genes and the realization that PHYB negatively regulates the resistance of rice to ShB via the suppression of ethylene signaling.

Soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) are the key regulators control trafficking of cargo proteins to their final destinations and plays key role in plant development; however, their roles in plant defense remain largely unknown. R-SNARE VAMP727 and Qa-SNARE SYP22 were previously reported to associate with vacuolar protein deposition and brassinosteroids (BRs) receptor BRI1 plasma membrane targeting. Here, we identified that VAMP727 and SYP22 are induced by infection of root-knot nematode (RKN), a plant pathogen, which cause severe growth defect and yield loss. Furthermore, decreased root-knot nematode (RKN) invasion, growth and disease index were observed in bri1-5 and SYP22ND, a SYP22 negative dominant mutants when compared to control plants. Overall, our results suggest that VAMP727-SYP22 SNARE complexes regulate plant defense might be via control of abundances of BRI1 on the plasma membrane.

The promotive effects of brassinosteroids (BRs) on plant growth and development have been widely investigated; however, it is not known whether BRs directly affect nutrient uptake. Here, we explored the possibility of a direct relationship between BRs and ammonium uptake via AMT1-type genes in rice (Oryza sativa). BR treatment increased the expression of AMT1;1 and AMT1;2, whereas in the mutant d61-1, which is defective in the BR-receptor gene BRI1, BR-dependent expression of these genes was suppressed. We then employed Related to ABI3/VP1-Like 1 (RAVL1), which is involved in BR homeostasis, to investigate BR-mediated AMT1 expression and its effect on NH4+ uptake in rice roots. AMT1;2 expression was lower in the ravl1 mutant, but higher in the RAVL1-overexpressing lines. EMSA and ChIP analyses showed that RAVL1 activates the expression of AMT1;2 by directly binding to E-box motifs in its promoter. Moreover, 15NH4+ uptake, cellular ammonium contents, and root responses to methyl-ammonium strongly depended on RAVL1 levels. Analysing AMT1;2 expression levels in different crosses between BRI1 and RAVL1 mutant and overexpression lines indicated that RAVL1 acts downstream of BRI1 in the regulation of AMT1;2. Thus, the present study shows how BRs may be involved in the transcriptional regulation of nutrient transporters to modulate their uptake capacity.

Nitrogen (N) is the most important macronutrient for plant growth and grain yields. For rice crops, nitrate and ammonium are the major N sources. To explore the genomic responses to ammonium supplements in rice roots, we used 17-day-old seedlings grown in the absence of external N that were then exposed to 0.5mM (NH4)2SO4 for 3h. Transcriptomic profiles were examined by microarray experiments. In all, 634 genes were up-regulated at least two-fold by the N-supplement when compared with expression in roots from untreated control plants. Gene Ontology (GO) enrichment analysis revealed that those upregulated genes are associated with 23 GO terms. Among them, metabolic processes for diverse amino acids (i.e., aspartate, threonine, tryptophan, glutamine, l-phenylalanine, and thiamin) as well as nitrogen compounds are highly over-represented, demonstrating that our selected genes are suitable for studying the N-response in roots. This enrichment analysis also indicated that nitrogen is closely linked to diverse transporter activities by primary metabolites, including proteins (amino acids), lipids, and carbohydrates, and is associated with carbohydrate catabolism and cell wall organization. Integration of results from omics analysis of metabolic pathways and transcriptome data using the MapMan tool suggested that the TCA cycle and pathway for mitochondrial electron transport are co-regulated when rice roots are exposed to ammonium. We also investigated the expression of N-responsive marker genes by performing a comparative analysis with root samples from plants grown under different NH4(+) treatments. The diverse responses to such treatment provide useful insight into the global changes related to the shift from an N-deficiency to an enhanced N-supply in rice, a model crop plant.

Abiotic stress, including salinity, drought and cold, severely affect diverse aspects of plant development and production. Rice is an important crop that does not acclimate to cold; therefore, it is relatively sensitive to low temperature stress. Dehydration-responsive element-binding protein 1s (DREB1s)/C-repeat binding factors (CBFs) are well known for their function in cold tolerance, but the transcriptional regulation of CBFs remains elusive, especially in rice. Here, we performed a yeast one-hybrid assay using the promoter of CBF1, a cold-induced gene, to isolate transcriptional regulators of CBF1. Among the seven candidates identified, an indeterminate domain (IDD) protein named ROC1 (a regulator of CBF1) was further analyzed. The ROC1 transcript was induced by exogenously-treated auxin, while it was not altered by cold or ABA stimuli. ROC1-GFP was localized at the nucleus, and ROC1 showed trans-activation activity in yeast. The electrophoretic mobility shift assay (EMSA) and ChIP analyses revealed that ROC1 directly bound to the promoter of CBF1. Furthermore, ROC1 mutants exhibited chilling-sensitive symptoms and inhibited cold-mediated induction of CBF1 and CBF3, indicating that ROC1 is a positive regulator of cold stress responses. Taken together, this study identified the CBF1 regulator, and the results are important for rice plant adaptation to chilling stress.

Highlight LPA1 suppresses auxin signalling that interacts with C-22-hydroxylated and 6-deoxo brassinosteroids, which regulates lamina inclination independently of OsBRI1., Lamina inclination is a key agronomical character that determines plant architecture and is sensitive to auxin and brassinosteroids (BRs). Loose Plant Architecture1 (LPA1) in rice (Oryza sativa) and its Arabidopsis homologues (SGR5/AtIDD15) have been reported to control plant architecture and auxin homeostasis. This study explores the role of LPA1 in determining lamina inclination in rice. LPA1 acts as a positive regulator to suppress lamina bending. Genetic and biochemical data indicate that LPA1 suppresses the auxin signalling that interacts with C-22-hydroxylated and 6-deoxo BRs, which regulates lamina inclination independently of OsBRI1. Mutant lpa1 plants are hypersensitive to indole-3-acetic acid (IAA) during the lamina inclination response, which is suppressed by the brassinazole (Brz) inhibitor of C-22 hydroxylase involved in BR synthesis. A strong synergic effect is detected between lpa1 and d2 (the defective mutant for catalysis of C-23-hydroxylated BRs) during IAA-mediated lamina inclination. No significant interaction between LPA1 and OsBRI1 was identified. The lpa1 mutant is sensitive to C-22-hydroxylated and 6-deoxo BRs in the d61-1 (rice BRI1 mutant) background. We present evidence verifying that two independent pathways function via either BRs or BRI1 to determine IAA-mediated lamina inclination in rice. RNA sequencing analysis and qRT-PCR indicate that LPA1 influences the expression of three OsPIN genes (OsPIN1a, OsPIN1c and OsPIN3a), which suggests that auxin flux might be an important factor in LPA1-mediated lamina inclination in rice.

Closely-located transposable elements (TEs) have been known to induce chromosomal breakage and rearrangements via alternative transposition. To study genome rearrangements in rice, an Ac/Ds system has been employed. This system comprises an immobile Ac element expressed under the control of CaMV 35S promoter, and a modified Ds element. A starter line carried Ac and a single copy of Ds at the OsRLG5 (Oryza sativa receptor-like gene 5). To enhance the transpositional activity, seed-derived calli were cultured and regenerated into plants. Among 270 lines regenerated from the starter, one line was selected that contained a pair of inversely-oriented Ds elements at the OsRLG5 (Oryza sativa receptor-like gene 5). The selected line was again subjected to tissue culture to obtain a regenerant population. Among 300 regenerated plants, 107 (36 %) contained chromosomal rearrangements including deletions, duplications, and inversions of various sizes. From 34 plants, transposition mechanisms leading to such genomic rearrangements were analyzed. The rearrangements were induced by sister chromatid transposition (SCT), homologous recombination (HR), and single chromatid transposition (SLCT). Among them, 22 events (65 %) were found to be transmitted to the next generation. These results demonstrate a great potential of tissue culture regeneration and the Ac/Ds system in understanding alternative transposition mechanisms and in developing chromosome engineering in plants.

Rhizoctonia solani AG1 IA is a soil-borne fungal phytopathogen that can significantly harm crops resulting in economic loss. This species overwinters in grass roots and diseased plants, and produces sclerotia that infect future crops. R. solani AG1 IA does not produce spores; therefore, understanding the molecular mechanism of sclerotia formation is important for crop disease control. To identify the genes involved in this process for the development of disease control targets, the transcriptomes of this species were determined at three important developmental stages (mycelium, sclerotial initiation, and sclerotial maturation) using an RNA-sequencing approach. A total of 5,016, 6,433, and 5,004 differentially expressed genes (DEGs) were identified in the sclerotial initiation vs. mycelial, sclerotial maturation vs. mycelial, and sclerotial maturation vs. sclerotial initiation stages, respectively. Moreover, gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) analyses showed that these DEGs were enriched in diverse categories, including oxidoreductase activity, carbohydrate metabolic process, and oxidation-reduction processes. A total of 12 DEGs were further verified using reverse transcription quantitative PCR. Among the genes examined, NADPH oxidase 1 (NOX1) and superoxide dismutase (SOD) were highly induced in the stages of sclerotial initiation and maturation. In addition, the highest reactive oxygen species (ROS) production levels were detected during sclerotial initiation, and enzyme activities of NOX1, SOD, and catalase (CAT) matched with the gene expression profiles. To further evaluate the role of ROS in sclerotial formation, R. solani AG1 IA was treated with the CAT inhibitor aminotriazole and H2O2, resulting in the early differentiation of sclerotia. Taken together, this study provides useful information toward understanding the molecular basis of R. solani AG1 IA sclerotial formation and maturation, and identified the important role of ROS in these processes.

Wheat stem rust caused by Puccinia graminis f. sp. tritici Eriks. & E. Henn. (Pgt), is a major disease that has been effectively controlled using resistance genes. The appearance and spread of Pgt races such as Ug99, TKTTF, and TTTTF, which are virulent to most stem rust-resistant genes currently deployed in wheat breeding programs, renewed the interest in breeding cultivars resistant to wheat stem rust. It is therefore important to investigate the levels of resistance or vulnerability of wheat cultivars to Pgt races. Resistance to Pgt races 21C3CTHQM, 34MKGQM, and 34C3RTGQM was evaluated in 136 Chinese wheat cultivars at the seedling stage. A total of 124 cultivars (91.2%) were resistant to the three races. Resistance genes Sr2, Sr24, Sr25, Sr26, Sr31, and Sr38 were analyzed using molecular markers closely linked to them, and 63 of the 136 wheat cultivars carried at least one of these genes: 21, 25, and 28 wheat cultivars likely carried Sr2, Sr31, and Sr38, respectively. Cultivars “Kehan 3” and “Jimai 22” likely carried Sr25. None of the cultivars carried Sr24 or Sr26. These cultivars with known stem rust resistance genes provide valuable genetic material for breeding resistant wheat cultivars.

Background Skin wound healing is a complex process that repairs multiple organ-tissues. Fibroblasts are key players of skin cells, whose migration is important during wound healing process. bFGF has shown a great efficacy to promote cell migration, but the precise mechanism by which bFGF regulates cell migration remains elusive. Objective The aim of this study was to find bFGF-regulated gene pools and further identify target molecules that participated in human fibroblast cell migration. Methods Skin primary fibroblasts and rat skin wound model were used to demonstrate the novel mechanism of bFGF regulating cell migration to accelerate wound healing. Cell migration was determined using the wound healing scratch assay. The differentially expressed genes and numerous biochemical pathways after bFGF treatment were identified by RNA-Seq analysis, and differentially expressed genes were further verified by qRT-PCR. siRNA duplex target to interfering the expression of PI3-kinase (p110α) was transformed into NIH/3T3 cells. Western blotting analysis was used to determine marker protein expressions. The invasive activity of fibroblasts was measured using 3D spheroid cell invasion assay. Results RNA-Seq analysis identified numerous biochemical pathways including the NF-κB pathway under the control of FGF signaling. bFGF negatively regulates the phosphorylation of IκB-α, the most well studied NF-κB signaling regulator while bFGF induces JNK phosphorylation. Application of Bay11-7082, a representative NF-κB inhibitor promoted cell migration, invasion and enhanced the JNKs phosphorylation. However, inhibition of JNKs blocked cell migration when NF-κB is inhibited. Moreover, application of the PI3 K inhibitor LY294002 together with Bay11-7082 maintained normal cell migration and knocking-down PI3 K (p110α) by a specific siRNA inhibited JNKs phosphorylation while maintaining normal IκBα phosphorylation, indicating that PI3 K and NF-κB signaling independently regulate JNKs activation. In addition, administration of bFGF or Bay11-7082 promoted rat skin wound repair and accelerated the invasion of fibroblasts. Conclusion This study sheds light on the mode of action of bFGF and identifies that the NF-κB-JNKs pathway is independent of the PI3K-JNKs pathway to accelerate fibroblast migration. In addition, bFGF and the relief of inflammation could be a favorable therapeutic approach for skin wound healing.