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Os02g0181300

2014-06-10T03:39:22Z

Hanweifang: /* Annotated Information */

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==Annotated Information==
===Function===
WRKY proteins constitute a family of mostly plant-specific zinc-finger transcription factors composed of 74 and 109 members in Arabidopsis and rice, respectively.A common feature of WRKY proteins is the WRKY domain, which is defined by a region of approximately 60 amino acids containing a conserved WRKYGQK amino acid sequence adjacent to a zinc-finger-like motif.Both regions are necessary for high-affinity binding to a canonical Wb-box motif containing a consensus sequence (C/T)TGAC(C/T). Based on primary structure, WRKY proteins have been classified into three groups (I, II, and III) and various subgroups (e.g., IIa, IIb, etc.)

== OsWRKY71 mRNA Levels Decline in Response to Exogenous GA Treatment ==
Analyses of available rice genome sequences reveals 77 OsWRKY genes. To investigate which of these genes are expressed in aleurone cells, RNA was
isolated from rice aleurone layers treated with no hormone, 1mM GA3, 20mM abscisic acid (ABA), or 1mM GA3 plus 20 mM ABA for 4 h, 24 h and 48 h. RT-PCR
was carried out with gene-speciﬁc primers (Supplemental Table I) for several OsWRKY genes, including OsWRKY71, OsWRKY77, and other control genes such
as RAmy3E, RAB21, and OsActin1, using the same set of RNA samples. Primers for OsWRKY71 were designed to amplify the entire coding region of this gene including all three introns. Hence, should a RNA preparation be contaminated with genomic DNA,a larger PCR fragment would be produced, as in the genomic DNA control. Clearly, none of the RNA samples was contaminated with a detec-able amount of genomic DNA. The mRNA level of OsWRKY71 is abundant in aleurone cells at the onset
of hormone treatments. Treatments without hormone or with GA3, ABA,and GA3 plus ABA for 4 h (Fig. 1, lanes 4–6) had little effect on the abundance of OsWRKY71 mRNA level.Prolonged GA3 treatments, for 24 h and 48 h , decreased the steady-state mRNA level of OsWRKY71 by 55% and 67%, respectively. As controls, the steady mRNA level of OsWRKY77 didn’t appear to be regulated by GA and that of RAmy3E,which encodes a GA-induciblea-amylase, was drastically enhanced in the same samples
treated with GA3 for 24 and 48 h. ABA or GA3 plus ABA treatments had little effect on the expression of the OsWRKY71 gene. By contrast,the mRNA level of RAB21, a reported ABA-inducible gene, increased greatly under the same ABA treatments. As expected, the mRNA levels of OsActin1,a constitutively expressed rice gene, were similar in all samples, suggesting that an equal amount of RNA was used for all RT-PCR reactions. These data show that OsWRKY71 is down-regulated by GA at either transcriptional and/or posttranscriptional levels. Alternatively, this mRNA could be under constant turnover,and reduction in its synthesis led to reduction of the mRNA level.

== OsWRKY71 Proteins Are Targeted to Nuclei of Aleurone Cells ==
To investigate the subcellular localization of OsWRKY71 protein, the cDNA sequence of the OsWRKY71 gene was fused in frame to the 39 end of the green ﬂuorescent protein (GFP) gene that is driven by the constitutive maize Ubiquitin (UBI) promoter. The fusion construct, UBI-GFP:OsWRKY71 and the control construct UBI-GFP were introduced, respectively, into barley aleurone cells by bombardment. Confocal microscopic observations indicated that GFP ﬂuorescence was detected in the
whole cell bombarded with the control construct UBIGFP. By contrast, the GFP:OsWRKY71 fusion protein was targeted to the nucleus. Aleurone cell nuclei were stained with SYTO17, which released red ﬂuorescence.

===Expression===
The WRKYssRNAi construct was introduced into Kitaake plants by Agrobacterium-mediated transformation. The presence of the transgene in individually transformed plants was confirmed by PCR. To determine the RNA levels of the endogenous OsWRKY IIa genes, real-time polymerase chain reaction (RT-PCR) was performed on RNA samples from 17 transgenic lines using gene-specific primers. Surprisingly, super-overexpression of all four genes was observed in nine transgenic lines when compared to the wild type controls. Four transgenic lines showed silencing of all four genes, called supersilencing, as expected, and four lines showed little change in gene expression relative to the controls. To ensure that the inserted construct is transcribed as expected, the gus linker expression was examined using RTPCR. As shown in Fig. 2, the gus linker expression was detected in all 17 transgenic lines.

===Evolution===
OsWRKY IIa subfamily members play both positive and negative regulatory roles in regulating plant innate immunity. Overexpression of OsWRKY62 compromises rice
basal defense and Xa21-mediated resistance to Xoo. By contrast, overexpression of OsWRKY71 results in elevated expression of defense-related genes and
enhanced resistance to Xoo. The work presented here demonstrates that super-overexpression of OsWRKY IIa subfamily members enhances basal defenseagainst Xoo. These data indicate functional interactions between OsWRKY IIa proteins in regulating plant innate immunity. Yeast two-hybrid and bimolecular fluorescence
complementation experiments provide support for physical interactions among the OsWRKYIIa proteins.
Similarly, Arabidopsis WRKY IIa subfamily members interact both physically and functionally in plant defense. AtWRKY IIa proteins form both homocomplexes and heterocomplexes. Association of different WRKY IIa members can alter their sequence-specific DNA binding activities. In addition, constitutive expression of
AtWRKY18 enhances resistance to P. syringae, while plants overexpressing AtWRKY18 with either AtWRKY40 or AtWRKY60 are more susceptible to both P. syringae and B.
cinerea. These observations indicate that association of different WRKY IIa members may alter defense gene regulation and ultimately affect the plant defense response.
A recent report on interactions of two transcriptional repressors and two transcriptional activators in modulating gibberellin signaling in aleurone cells provides an intriguing example of how different transcription factor complex affects the expression of target gene. HvWRKY38,a barley ortholog of OsWRKY71, functions as a repressor of GA-induced expression of Amy32b a-amylase. When present individually, the inductive activities of SAD, a DNA-binding with one finger (DOF) protein and HvGAMYB (a R2R3 MYB protein) on Amy32b expression are blocked by HvWRKY38. However, SAD and HvGAMYB together overcome the inhibitory effect of HvWRKY38. Interestingly,the synergistic effect of SAD and HvGAMYB transcriptional activators is almost abolished when repressor BPBF, a barley prolamin-box binding factor which is another DOF protein, is present with HvWRKY38. Further investigation shows that HvWRKY38 interacts with HvBPBF in the nuclei of aleurone cells. Moreover, HvWRKY38 interferes with the binding of HvGAMYB to the Amy32b promoter. Taken together, these studies suggest that relative amounts of activator and repressor proteins in a complex can modulate the expression of target genes.
OsWRKY IIa proteins contain putative leucine zipper motifs at the N-terminus, which suggests potential homodimerization or heterodimerization between proteins. It has been shown that leucine zipper motifs are critical for the physical interaction of AtWRKY IIa proteins and homodimerization of HvWRKY38. OsWRKY71 has also recently been reported to function as a transcriptional repressor.As a positive regulator of plant defense response,OsWRKY71 may play an indirect role in transcriptional activation of defense-related gene expression by interacting with other regulatory proteins. Taken together, we hypothesize that different combinatorial dimers formed by OsWRKY IIa proteins may exhibit different functions in regulating target gene expression. Moreover, changes in the participants within protein complexes caused by mutation,overexpression or environmental stress can also affect the expression of target genes, leading to altered plant defense response. This may explain the data observed with rice lines overexpressing OsWRKY62 alone (susceptible) compared with plants overexpressing all four OsWRKY IIa proteins (resistant).
While ineffective gene silencing by short interference RNA (siRNA) is commonly observed, it has not previously been observed that a siRNA construct can lead to super-overexpression of the target genes. Inverted repeats are usually found to be very efficient for silencing, although different levels of efficiency of
silencing are not uncommon (Chern et al. 2005a). We have searched the literature for an explanation of the superoverexpression phenomenon and found reports of RNAs that are able to stimulate transcription; however, these examples are directed toward promoter sequences, not the translated region of a gene. For instance, Dahiya and associates discovered that exogenous dsRNAs designed against human promoter regions increase expression of the corresponding coding region. Our results indicate that simultaneous over-expression of all the members of the rice WRKY IIa complex reveals a previously unobserved outcome of attempted gene silencing. Although the mechanism remains unknown at this point, one possibility is that self-regulation by WRKY genes or regulation via other transcription factors and proteins may contribute to this phenotype.

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==Labs working on this gene==
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==References==
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==Structured Information==
{{JaponicaGene|
GeneName = Os02g0181300|
Description = WRKY transcription factor 71 (Transcription factor WRKY09)|
Version = NM_001052629.1 GI:115444628 GeneID:4328512|
Length = 1393 bp|
Definition = Oryza sativa Japonica Group Os02g0181300, complete gene.|
Source = Oryza sativa Japonica Group

ORGANISM Oryza sativa Japonica Group
Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
Spermatophyta; Magnoliophyta; Liliopsida; Poales; Poaceae; BEP
clade; Ehrhartoideae; Oryzeae; Oryza.
|
Chromosome = [[:category:Japonica Chromosome 2|Chromosome 2]]|
AP = Chromosome 2:4543031..4544423|
CDS = 4543031..4543177,4543287..4543709,4543836..4543952,4544064..4544423|
GCID = <gbrowseImage1>
name=NC_008395:4543031..4544423
source=RiceChromosome02
preset=GeneLocation
</gbrowseImage1>|
GSID = <gbrowseImage2>
name=NC_008395:4543031..4544423
source=RiceChromosome02
preset=GeneLocation
</gbrowseImage2>|
CDNA = <cdnaseq>atggatccgtggattagcacccagccttcgctgagcctggacctccgcgtcgggctgccggcgacggcggccgtcgccatggttaagcccaaggtgctcgtcgaggaggacttctttcaccagcagcctctcaagaaagacccagaggttgcggcgctggaggcggagctgaagcggatgggcgcggagaaccggcagctgagcgagatgctggcggcggtggcggccaagtacgaggcgctgcagagccagttcagcgacatggtcaccgccagcgccaacaacggcggcggcggcggcaacaacccgtcgtccacctccgagggcggctccgtctcgccgtcgaggaagcgcaagagcgagagcctcgacgactccccgccgccgccgccgccgccgcacccacacgcggcgccgcaccacatgcacgtcatgcccggcgccgccgccgccggctacgccgaccagaccgagtgcacctccggcgagccctgcaagcgcatccgcgaggagtgcaagcccaagatctccaagctctacgtccacgccgacccatccgacctcagcctggtggtgaaagatgggtaccaatggaggaagtatggtcagaaggtcaccaaggacaacccctgcccaagagcctacttcagatgctcatttgctcccgcctgccctgtcaagaagaaggttcagagaagcgcggaggacaacacgatcctcgtggcgacgtacgagggggagcacaaccacggccagccgccgccgccgctgcagtcggcggcgcagaacagcgacggctccggcaagagcgccgggaagccaccccatgcgccggcggcggcgccgccggcgccggtggtgccgcaccgtcagcacgaaccggtcgtcgtcaacggcgagcagcaggccgcggcggcgtcggagatgatcaggcggaacctggccgagcagatggcgatgacgctgacgcgtgacccaagcttcaaggcggcgctcgtcaccgccctctccggccgcatcctcgagctctcgccgaccaaggattga</cdnaseq>|
AA = <aaseq>MDPWISTQPSLSLDLRVGLPATAAVAMVKPKVLVEEDFFHQQPL KKDPEVAALEAELKRMGAENRQLSEMLAAVAAKYEALQSQFSDMVTASANNGGGGGNN PSSTSEGGSVSPSRKRKSESLDDSPPPPPPPHPHAAPHHMHVMPGAAAAGYADQTECT SGEPCKRIREECKPKISKLYVHADPSDLSLVVKDGYQWRKYGQKVTKDNPCPRAYFRC SFAPACPVKKKVQRSAEDNTILVATYEGEHNHGQPPPPLQSAAQNSDGSGKSAGKPPH APAAAPPAPVVPHRQHEPVVVNGEQQAAAASEMIRRNLAEQMAMTLTRDPSFKAALVT ALSGRILELSPTKD</aaseq>|
DNA = <dnaseqindica>1..147#257..679#806..922#1034..1393#atggatccgtggattagcacccagccttcgctgagcctggacctccgcgtcgggctgccggcgacggcggccgtcgccatggttaagcccaaggtgctcgtcgaggaggacttctttcaccagcagcctctcaagaaagacccagaggtaggtcatgtgattaagcatacgtgcacggacgatcgacggccgccatggctgggagcgtgatgagcttaatttcttgagctgatttggggttttttttgttgtgaaggttgcggcgctggaggcggagctgaagcggatgggcgcggagaaccggcagctgagcgagatgctggcggcggtggcggccaagtacgaggcgctgcagagccagttcagcgacatggtcaccgccagcgccaacaacggcggcggcggcggcaacaacccgtcgtccacctccgagggcggctccgtctcgccgtcgaggaagcgcaagagcgagagcctcgacgactccccgccgccgccgccgccgccgcacccacacgcggcgccgcaccacatgcacgtcatgcccggcgccgccgccgccggctacgccgaccagaccgagtgcacctccggcgagccctgcaagcgcatccgcgaggagtgcaagcccaagatctccaagctctacgtccacgccgacccatccgacctcagcctggtacgaatccccaaccccaattaacatccgatcatctcatttcatgtgcctaacttaacttctttttgttctgaattctagccttgattaattaattaatcctgttcttgcaatgaatccatgcaggtggtgaaagatgggtaccaatggaggaagtatggtcagaaggtcaccaaggacaacccctgcccaagagcctacttcagatgctcatttgctcccgcctgccctgtcaagaagaaggtaacttaaattcctctaactaattccaaagtgatttcgtaagttttgctaaatttagtgcaaattcgagcctgtttctgaactttgttcgtggttcaaatgttgtttcaggttcagagaagcgcggaggacaacacgatcctcgtggcgacgtacgagggggagcacaaccacggccagccgccgccgccgctgcagtcggcggcgcagaacagcgacggctccggcaagagcgccgggaagccaccccatgcgccggcggcggcgccgccggcgccggtggtgccgcaccgtcagcacgaaccggtcgtcgtcaacggcgagcagcaggccgcggcggcgtcggagatgatcaggcggaacctggccgagcagatggcgatgacgctgacgcgtgacccaagcttcaaggcggcgctcgtcaccgccctctccggccgcatcctcgagctctcgccgaccaaggattga</dnaseqindica>|
Link = [http://www.ncbi.nlm.nih.gov/nuccore/NM_001052629.1 RefSeq:Os02g0181300]|
}}
[[Category:Genes]]
[[Category:Japonica mRNA]]
[[Category:Oryza Sativa Japonica Group]]
[[Category:Japonica Genes]]
[[Category:Japonica Chromosome 2]]
[[Category:Chromosome 2]]

Os02g0181300

2014-06-09T17:10:30Z

Hanweifang: /* Expression */

Os02g0181300

2014-06-09T16:55:43Z

Hanweifang: /* Annotated Information */

Os02g0181300

2014-06-09T16:34:15Z

Hanweifang: /* Function */