Research Progress on Functional Analysis of Rice WRKY Genes Rice Science, 17: 60-72.

Rice (Oryza sativa) is the first monocot plant to have its genome sequenced, and has become a model for the genomic study of grass species. The finished quality sequence showed that rice had the genome size of 389 Mb ^[1]. Approximately 1600 transcription factor (TF) genes in rice were identified ^[2], and several large TF families included more than 100 members, such as MYB, WRKY, AP2/EREBP, bHLH, bZIP, C₂H₂ and NAC ^[3]. Although their precise regulatory functions are largely unknown, the fact that most of these TFs in Arabidopsis appear to be functional suggests that they also play important roles during the disparate biological requirements in rice.

for binding affinity of WRKY TFs to the consensus cis-acting elements W box 5'-C/TTGACT/C-3' and become the basis for classifying WRKY TFs into three groups ^[5]. Further studies revealed that the binding affinity also depended on additional adjoining DNA sequences outside the TGAC core sequences ^[6-7].

Searching the sequence of the rice genomic databases, several teams had found a total of 102 copies of WRKY genes in indica rice and 103 putative rice genes encoding the WRKY domain in japonica rice ^[8-9]. The first OsWRKY cDNA, OsWRKY04 (denoted as OsWRKY1 by Kim et al ^[10]), was cloned in rice upon infection by the fungal blast pathogen Magnaporthe grisea. Another gene, OsWRKY12, was reported to be induced by the bacterial blight pathogen Xanthomonas oryzae pv. Oryzae (Xoo) as OsiWRKY ^[11] and responded to Xoo inoculation, wounding, etc. as OsWRKY03 ^[12] (In this review, we accept the fact that OsWRKY12 was renamed as OsWRKY03 for its reported biology function). It seemed that the nomenclature of WRKY genes used in rice was confusing. Among all the rice WRKY genes, the expression profiles of 70 were reported, the function of eight was traversed in homologous transgenic rice, five analyzed in heterologous transgenic Arabidopsis, and four checked using transient expression assay in barley aleurone cells.

An increasing number of studies in rice have convincingly established the importance of WRKY TFs in transcriptional reprogramming during its immune response. For instance, overexpression of OsWRKY13 enhanced rice resistance to Xoo and M. grisea ^[13-14]. Similarly, ectopic expression of OsWRKY45 and OsWRKY31 genes resulted in enhanced resistance to fungal blast but overexpressions of the genes OsWRKY19, 62 and 76 did not ^[15-16]. Simple analysis showed that OsWRKY53 and OsWRKY71 were considered to take part in elicitor-induced defense signaling pathways in rice ^[17-18]. The expression of OsWRKY03, 13 and 62 were rapidly induced in incompatible host-pathogen interaction as compared to compatible interaction ^[19], but their regulating functions seemed to be different ^{[12, 14, 20]}. Heterologous analysis of OsWRKY23 and OsWRKY33 in transgenic Arabidopsis indicated that these two genes could activate the transcription of PR1 gene ^[21-22]. Finally, overexpressed OsWRKY89 showed enhanced resistance against fungal blast and the white-backed plant hopper (Sogatella furcifera) ^[23]. These results show that rice WRKY transcription factors are widely involved in the biotic defense responses.

Beside biotic stresses, rice WRKY genes are also induced or suppressed by various abiotic stresses, even play roles in plant development and senescence. To seek the function of rice WRKY genes under various abiotic stresses, we had previously screened 13 WRKY genes in rice whose expression profiles exhibited great differences in both the pattern and timing of their responses to different abiotic treatments ^[24]. Furthermore, our ectopic overexpression of OsWRKY08 ^[25] and OsWRKY45 ^[26] transgenic Arabidopsis showed enhanced tolerance to drought stress and osmotic stress, respectively. Moreover, overexpression of OsWRKY89 and OsWRKY11 genes altered the UV-B irradiation tolerance and dry heat tolerance of transgenic rice, respectively ^{[23, 27]}. The semi-quantitative RT-PCR analysis showed that OsWRKY17, 23, and 82 were induced in senescing rice leaves ^{[21, 28-29]}, and overexpression of OsWRKY23 gene enhanced susceptibility to dark-induced senescence in transgenic Arabidopsis ^[21]. In addition, heterologous expression of OsWRKY72 gene weakened apical dominance of transgenic Arabidopsis and homologous expression of OsWRKY31 gene

reduced lateral root formation and elongation of transgenic rice ^{[16, 30]}. This review aims at bringing the sufficient developments in biological function of WRKY transcription factors in rice in the past decade.

According to recent data from DRTF, there are 111 and 129 gene models in indica and japonica rice, respectively ^[31]. After Shen et al ^[32] identified 77 WRKY genes in both indica and japonica rice, we ferreted an additional 24 novel WRKY genes out of the complete rice genome and increased the number of WRKY genes to 97 in 2004 ^[24]. In 2005, Shen’s laboratory ^[33], Guo’s laboratory ^[34], and the Samuel Roberts Noble Foundation ^[35] predicted 81, 102 and 109 WRKY genes respectively in the rice genome. Recently, Ross et al ^[8] defined 102 putative WRKY genes in indica rice and Ramamoorthy et al ^[9] confirmed 103 copies of WRKY genes in japonica rice, which had been generally accepted.

As the rule of Eulgem, WRKY family members are divided into three groups based on the number of WRKY domains and the zinc finger-like motif ^[5]. Recent NMR solution and crystal structure forecasts revealed that the C-terminal WRKY domain consists of a four- or five-stranded β-sheet and with the N-terminal Cys₂His₂ or Cys₂HisCys residues form a zinc-binding pocket ^[36-37]. More noticeably, careful comparison of rice WRKY proteins with Arabidopsis WRKY proteins showed that several novel WRKY proteins whose WRKY domain sequence is WRKYGEK exist in rice but not in Arabidopsis ^[24].

Since there are more WRKY genes and several independent research groups are working on these genes, the nomenclature of WRKY genes in rice is complex. In addition to our laboratory and Shen’s laboratory using the same system, there are four main labs using three kinds of nomenclatures ^{[24, 32-35]}. Therefore, it appears that the same gene may has three names at least. For example, we cloned OsWRKY08 ^[24], which was named as OsWRKY61 by Wu et al ^[34] and denoted as OsWRKY44 by Zhang and Wang ^[35]; Another gene OsWRKY72^[30], located on chromosome 11, was designated to the locus number LOC_Os11g29870 (TIGR) in our reference, however, this nomenclature was referred to a gene mapped on chromosome 7 by Wu et al ^[34] and chromosome 3 by Zhang and Wang ^[35]. Several other research groups also named their target WRKY genes by themselves, such as OsWRKY04 and OsWRKY12 ^[24], which are known as OsWRKY01 and OsWRKY03 by Kim et al ^[10] and Liu et al ^[12], respectively. Additionally, a few authors made mistakes when citing the gene name. For example, OsiWRKY ^[11] was regarded as OsWRKY80 by Ryu et al ^[19], in fact it should be named OsWRKY12 ^[8]. It seemed that nomenclature of WRKY genes in rice was confusing.

In light of current trends, many reports are adopting the nomenclature of Shen’s and ours, and a couple of articles use Guo’s nomenclature, while the nomenclature of Zhang and Wang is abandoned. We encourage the use of the Shen’s and our designations in future studies, and follow that three genes’ name OsWRKY03, OsWRKY31 and OsWRKY89 transform to OsWRKY12, OsWRKY55 and OsWRKY100 as listed in Table 1, respectively. Our nomenclature creates the number to name random genes following the priority order while Guo’s nomenclature as the chromosomal localization orders in general ^[34]. Over 20 WRKY gene names (>20%) of Guo’s nomenclature breaks the rule themselves as the chromosomal localization order such as their OsWRKY96, 97, 98, 99 and 100 ^[34], and there are some newly identified WRKY genes whose names would violate the rules time after time such as Os04g04300 and Os08g09840. These two novel WRKY genes in rice are named as OsWRKY101 and OsWRKY102 in Table 1.

Gene expression analysis is essential for understanding the probable biological function. In Arabidopsis, 49 out of 74 tested WRKY genes were induced or suppressed by bacterial infection or salicylic acid treatment ^[19], and a lot of them were validated to play roles in defense signaling. Up to now, five primary teams had performed relative systematic analysis of OsWRKY gene expression in rice under abiotic stresses, and in SA treated, M. grisea-infected and Xoo-inoculated rice plants.

WRKY proteins involved in defense against attacks by plant pathogens had been identified in Arabidopsis. Ryu et al ^[19] reported that the genes OsWRKY07, 10, 11, 30, 32, 45, 62, 64, 67, 70, 76, 82, 83, 84, and 85 were remarkably induced by an incompatible interaction between M. grisea and rice. The expression of three (OsWRKY45, 62, and 76) out of these genes was decreased, and except OsWRKY64, 82 and 84, the expression of other nine WRKY genes was increased upon the bacterial pathogen Xoo in rice plants. Gloria’s microarray data showed that the transcripts of OsWRKY23, 28, 45, 56, and 72 were increased more than 5-fold at early stage under a compatible interaction between M. grisea and rice ^[38]. Guo’s laboratory informed that OsWRKY03 and OsWRKY70 were induced by Xoo and M. grisea, respectively ^{[11, 39]}. RNA gel blot hybridization analysis by Shimono et al ^{[15, 40]} displayed that OsWRKY45, 62 and 76 were up-regulated within 3 h after BTH application but OsWRKY19 was not. Microarray data by Shimizu et al ^[41] revealed that the transcripts of OsWRKY08, 55 and 77 were up-regulated more than 2-fold in rice plants infected with rice dwarf virus. Swarbrick et al ^[42] screened OsWRKY19, 40, 45, 62, 76, 77 and 90, which were significantly up-regulated (>5-fold) only in the roots of the rice variety Nipponbare undergoing a resistant interaction with Striga hermonthica. From suspension-cultured rice cells treated with a chitin oligosaccharide elictor, Chujo et al ^[43] identified two WRKY genes OsWRKY53 and OsWRKY71.

Seki et al ^[44] screened several WRKY genes respond to abiotic stresses in Arabidopsis. Afterwards, Qiu et al ^[24] analyzed the expression of OsWRKY08, 09, 12, 13, 14, 16, 17, 21, 23, 24, 26, 30 and 45 after treating with NaCl, PEG, cold and heat. Previous studies indicated that the expression of OsWRKY03 and OsWRKY89 was induced upon mechanical wounding ^{[11, 45]}. Ramamoorthy et al ^[9] performed systematic analysis of 65 WRKY genes expression under abiotic (salinity, drought and cold) stresses and in five phytohormone (ABA, IAA, GA₃, MeJA and SA)

treated rice plants. Ryu et al ^[19] also showed the transcripts of OsWRKY45 and OsWRKY62 were increased in SA-treated leaves, while jasmonic acid increased the transcripts of OsWRKY10, 82 and 85, moreover, OsWRKY30 and OsWRKY83 responded to both SA and JA treatments. Liu et al ^[46] isolated the genes OsWRKY01, 12, 15, 24, 42, 53, 69, 71, 74 and 93 from rice seedlings treated with SA. Shimono et al ^[15] identified OsWRKY19, 45, 62 and 76 as BTH- responsive genes in their microarray data. In rice aleurone cells, Xie et al ^[33] carried out Northern-blot analyses and showed that the transcripts of OsWRKY24, 51, 71 and 72 were enhanced upon ABA treatment, while the mRNA levels of OsWRKY51 and OsWRKY71 were down-regulated in response to GA3 treatments. In addition, OsWRKY08, 72 and 31 were induced in auxin analogues treated rice seedlings ^{[16, 25, 30]}.

The roles of WRKY genes in Arabidopsis had been reviewed several times ^{[5, 47-49]}, and Ross et al ^[8] had reviewed the studies performed in monocotyledonous plants between 2003 and 2006. The objective of this part is to comprehensively review the progress in the function of rice WRKY TFs.

Plants resist the attacks of harmful microorganisms and insects through multiple mechanisms including basal defense, PAMP-triggered immunity (PTI), effector-triggered immunity (ETI), and systemic acquired resistance (SAR) ^[48]. Global expression profiling revealed that the major difference among these defense responses was quantitative or temporal rather than qualitative ^[50]. It seemed that most pathogens trigger an interconnected plant signaling network. Indeed, multiple studies had revealed that WRKY proteins of Arabidopsis regulated transcriptional reprogramming and formed a web ^[48]. Both in Arabidopsis and rice, the majority of WRKY genes responded to pathogen infection or SA/JA treatment ^{[19, 47]}. The most devastating diseases of rice worldwide were bacterial blight caused by Xoo and fungal blast caused by M. grisea, which induced the expression of dozens of OsWRKY genes ^[19]. Functional analysis showed that these WRKY members had been associated with pathogen defense in rice.

Transcript levels of OsWRKY03 and OsWRKY71 were strongly up-regulated by pathogen-mimicking stimuli ^{[12, 46]}. OsWRKY03-expressing rice plants had enhanced expression of peroxidase gene, phenylalanine ammonia-lyase gene, PR1b and NH1 ^[12]. In OsWRKY71 overexpressing rice, the transcript levels of NH1 and PR1 were also higher than those in wild-type plants and the resistance to virulent Xoo was enhanced ^[46]. Thus, it could be seen that OsWRKY03 and OsWRKY71 genes functioned upstream of OsNH1 in defense signaling. Different from OsWRKY71, OsWRKY45 was neither downstream nor upstream of NH1, and gene function analysis suggested that WRKY45 was involved in a pathway independent of NH1 downstream of SA and played a positive role in improving resistance against M. grisea disease ^{[15, 40]}. Microarray analysis from Yamane’s laboratory reported that both OsWRKY53 and OsWRKY71 had been identified as chitin oligosaccharide elicitor-induced genes from suspension-cultured rice cells ^[17-18]; In rice cells overexpressing OsWRKY71, six defense-related chitinase genes were significantly up-regulated ^[17]; And in the rice plants overexpressing OsWRKY53, six genes PBZ1, PR-14, Chitinase 1, PR-5, Chitinase 2, and peroxidase gene were also significantly up-regulated ^[18]. These results indicated that the function of OsWRKY53 and OsWRKY71 referred to PTI. Moreover, both OsWRKY62 and OsWRKY13 were recently discovered to be negative regulators of ETI ^{[20, 51]}. The rice genes Xa3 and Xa21 conferred race-specific resistance to Xoo. Xa21 was observed to bind to OsWRKY62, and the overexpression of OsWRKY62 suppressed the activation of defense related genes Betv1, PR1a, PR10 and PBZ1 ^[20]. However, the overexpression of OsWRKY62 and OsWRKY76 had no effect on resistance to a compatible race of M. grisea ^[15]. The expression of Xa3 was rapidly induced with OsWRKY13 and OsNH1, and their ecotopic overexpressor lines analysis suggested that OsWRKY13 and OsNH1 were involved in R gene-mediated resistance against Xoo ^[51]. In addition, OsWRKY89 overexpressing rice showed enhanced resistance against fungal blast and the white-backed plant hopper Sogatella furcifera, and OsWRKY89 knockdown lines displayed increased susceptibility to M. grisea ^[23]. OsWRKY31 overexpressing rice displayed enhanced resistance to fungal blast, and OsWRKY31 RNA interference lines showed no altered disease phenotype ^[16]. Finally, the assays of transient coexpression of OsWRKY33 and OsBWMK1 in Arabidopsis protoplasts suggested that OsWRKY33 might mediate SA-dependent defense responses ^[22].

The WRKY gene family in Arabidopsis was recently suggested to play a key role in the response of plants to abiotic and oxidative stresses ^[52]. The majority types of abiotic stresses such as heat, cold, flooding, drought, salinity, high-light and UV-B radiation stresses disrupt the metabolic balance of cells, resulting in enhanced accumulation of reactive oxygen species (ROS) and activation of ABA pools ^[52-53]. In Arabidopsis, the transcript levels of AtWRKY25 and AtWRKY33 were enhanced by salinity, and their ectopic expression plants were sufficient to increase Arabidopsis NaCl tolerance, while enhancing sensitivity to ABA ^[54]. After screening several WRKY genes from rice induced by abiotic stresses ^[24], we generated transgenic Arabidopsis plants that overexpressed OsWRKY08, 45 and 72 genes under the CaMV35S promoter. Functional analysis of these transgenic plants showed that over-expression of OsWRKY45 enhanced salt and drought tolerance in Arabidopsis ^[26], heterologous expression of OsWRKY08 improved the osmotic stress tolerance in Arabidopsis ^[25], and constitutive expression of OsWRKY72 affected root growth and related stress tolerance (Fig. 1). Among these three kinds of transgenic Arabidopsis, the analysis results of downstream gene expression suggested that OsWRKY45 ^[26] and OsWRKY72 (unpublished data) could activate the ABA-dependent abiotic stress response genes but OsWRKY08 ^[25] could not. Furthermore, OsWRKY89 overexpression also seemed to enhance tolerance to UV-B irradiation by regulating the wax content/deposition on the leaf surface of transgenic rice ^[23], overexpressing OsWRKY11 enhanced dry heat tolerance in transgenic rice seedlings ^[27], constitutive expression of OsWRKY13 suppressed salt and cold defense responses via mediating redox homestasis ^[55], and overexpression of OsWRKY45 could enhance rice tolerance to drought without causing deleterious fertile effects ^[56]. Thus, these WRKY genes might be useful for breeding varieties with multiple resistances to meet the needs of

global rice production.

Leaf senescence is considered the last stage of leaf development, in which mature cells, tissues or sometimes the entire leaf organs go through a series of programmed cell death (PCD) processes. In addition to age, leaf senescence can also be mediated by both environmental and genetic factors. Expression profiling in Arabidopsis revealed that the transcripts of many WRKY genes were significantly up-regulated during leaf senescence and the WRKY proteins constituted the second largest group of transcription factors of the senescence transcriptome ^[57]. Biological function analysis of AtWRKY6 ^[58], 53 ^[59] and 70 ^[60] revealed that individual WRKY factors took part in the genetic web of leaf senescence. For rice, the expression of OsWRKY4 and OsWRKY82 was up-regulated in flag leaf natural early senescence process ^[29], and the transcript of OsWRKY17 gene was up-regulated in the response of rice roots to Fe-deficiency stress and subsequent senescence ^[28]. Furthermore, we found that the overexpression of OsWRKY23 accelerated leaf senescence in darkness, and two senescence-associated marker genes SAG12 and SEN1 were altered in 35S::OsWRKY23 lines ^[21]. These results suggested that OsWRKY23 was a novel modulator of dark-induced leaf senescence.

Starch is deposited in the endosperm as granules, and its synthesis and deposition in the endosperm depend on a dozen enzymes in plant sugar signaling. Molecular analysis showed that five different types of cis-elements had been identified in sugar-regulated plant promoters: SP8, SURE, TGGACGG, G-box, and B-box ^[61]. The first identified WRKY protein in sweet potato, SPF1, bound to SP8 elements upstream of the sugar-responsive sporamin and β-amylase genes whose expression was induced by sugars that function as a repressor ^[62]. In barley, a WRKY transcription factor SUSIBA2 was involved in sugar-mediated regulation of starch synthesis through binding to the SURE elements of iso1 promoter as an activator ^[61]. In rice cells under sucrose-starved stress, the expression of OsWRKY62, 67, 45 and 72 was up-regulated ^[63]. Also in barley aleurone cells, another WRKY factor HvWRKY38 blocked GA-induced expression of Amy32b via binding to Amy32b promoter ^[64]. OsWRKY71, the ortholog of HvWRKY38, could interact with itself and OsWRKY51, bound to W boxes in the Amy32b promoter, and further affected seed germination and post-germination growth through activating starch degradation in aleurone cells ^[65]. Using a transient expression experiments, Shen’s team demonstrated that ABA-inducible and GA-repressible rice WRKY genes OsWRKY51 and OsWRKY71 mediated the cross-talk of GA and ABA signaling, OsWRKY24 inhibited both GA and ABA signaling, OsWRKY24 and OsWRKY45 regulated the ABA-inducible HVA22 promoter in a negative manner, and OsWRKY72 and OsWRKY77 regulated the ABA- inducible HVA22 promoter in a positive manner ^[8].

To some extent, plant architecture is influenced by such factors as nutrition, light, temperature, humidity and plant density. However, plant architecture is also dominated by its own genetic program. In Arabidopsis, wrky44 knockout mutants and WRKY75 RNAi mutants showed the unbranched and decreased number of trichomes and increased number of lateral roots and root hairs, respectively ^[66-67]. For OsWRKY72, its overexpression in Arabidopsis also showed reduced lateral roots, and specially displayed more branches than wild type ^[30]. The phenotype of both 35S::OsWRKY72 and 35S::AtWRKY6 were dwarf with partially necrotic leaves, early flowering, and reduction in their apical dominance, which suggested that the overexpression of these two genes might interfere in the auxin signaling pathway ^[58]. Furthermore, the changed phenotype of root formation also existed in transgenic rice overexpressing OsWRKY31, and two early auxin-response genes were constitutively expressed in transgenic rice seedlings ^[20].

In both basal defense and SAR, the accumulation of defense hormone SA or JA and transcriptional cofactor NPR1 (Nonexpresser of pathogenesis-related genes 1) dominate the activation of transcriptional network ^[68]. Intriguingly, some WRKY proteins mediate NPR1 expression, while some are operated by NPR1. WRKY and NPR1 are important node proteins in SA-dependent defense signaling pathway and have been implicated in SA/JA cross talk ^[47]. In Arabidopsis, the overexpression of AtWRKY70 caused up-regulated transcript of SA-responsive PR genes and suppressed the expression of methyl jasmonate (MeJA)-induced PDF1.2 gene ^[60]. In a double mutant in which AtWRKY11 and AtWRKY17 were knocked out, the transcripts of JA-responsive genes decreased to lower levels, whereas those of SA-responsive genes were notably higher ^[69]. And the expression of AtWRKY62 was strongly induced by SA and JA in wild type, but not in mutant npr1-3 ^[70]. In rice, the overexpression of cytosolic OsNH1 (ortholog of AtNPR1) down regulated JA-responsive transcription and played a similar role of AtNPR1 ^[71]. OsWRKY13 had been implicated in activating SA-biosynthesis and SA-response genes while suppressing JA signaling. Not only could it bind to the promoters of OsAOS2 and OsLOX genes involved in JA synthesis in defense response, it also bound to the promoter of PR1a gene functioning in SA-dependent pathway ^[72]. Moreover, OsWRKY03 and OsWRKY71 functioned upstream of OsNH1 in defense signaling ^{[12, 46]}; OsWRKY45 appeared to mediate SA signaling but independent of NH1 ^[15], and regulated the expression of PR genes as OsWRKY13, 53 and 62 ^{[13, 17, 20]}. In addition, OsWRKY71 and OsWRKY24 had been shown to bind to the promoter of Amy32b ^{[65, 73]}. A rice stemar-13-ene synthase gene OsDTC2, whose promoter contained six W box motifs ^[74], was probably activated by WRKY proteins in rice.

An increasing number of studies have strongly confirmed that several WRKY genes can be subject to auto/cross-regulation by themselves or other WRKYs. PcWRKY1 activated a reporter gene driven by the promoters of three potential target genes PcPR1, PcPR10 and PcWRKY1 ^[48]. AtWRKY18, 40, and 60 interacted with themselves and with each other to form complexes ^[75]. AtWRKY11 and AtWRKY17 negatively regulated the expression of AtWRKY70 and AtWRKY54 ^[69]. NaWRKY6 transcript accumulation was shown to be dependent on NaWRKY3 expression ^[76]. In rice, gel mobility shift assays showed that OsWRKY13 could bind to its own promoter, and other gene expression analysis in OsWRKY13-activated lines showed that the transcript levels of OsWRKY10 and OsWRKY68 were significantly higher than that in wild type, and the transcript levels of OsWRKY14, 24, 42, 45 and 71 were significantly lower than those in wild type ^[72]. In the region from –514 to –251 bp of OsWRKY53 promoter, there were three W-boxes which were indicated to be involved in regulation of the elicitor- responsiveness or basal promoter activity of OsWRKY53 through analysis of mutants fused to FLUC ^[77].

Sequence analysis of WRKY proteins showed that dozens of WRKY TFs contained D motif (LSPSNLLESPxL), which was a potential phosphorylation sites for MAP kinases ^[5]. In tobacco, SIPK-induced direct phosphorylation of NtWRKY1 enhanced its DNA-binding activity to the W-box sequence ^[78]. In Arabidopsis, AtWRKY22 and AtWRKY29 had been identified as essential downstream components of MAP3/MPK6 cascades ^[79]. MEKK1 was also shown to phosphorylate AtWRKY53 ^[80]. An MPK4 substrate MKS1 was demonstrated to interact with AtWRKY33, which could be phosphorylated by MPK4 ^[81]. In rice, heterologous expression of OsBWMK1 increased PR gene expression and induced HR-like cell death like SIPK, and yeast-two hybrid screen displayed that OsBWMK1 could phosphorylate OsWRKY33 ^[22]. These imply that defence-induced MAPK signaling cascades are associated with specific WRKY factors, and implicate the up-stream events of plant defense signaling. Finally, Cai et al ^[14] identified two novel pathogen-responsive cis-acting elements in the promoter of OsWRKY13 which was bound by six proteins including OsWRKY13 itself. This evidence indicates that the expression of OsWRKY13 in response to pathogen invasion may be regulated by multiple factors.

Overwhelming majority of WRKY TFs is widely present in all plants. Since the phylogenetic trees among the Arabidopsis WRKY genes were reported, the phylogenetic relationship of WRKY genes had been constructed in moss, ferns, rice, tobacco, barley, cowpea, soybean, papaya, poplar and sorghum ^[25]. WRKY TFs are not restricted to the plant kingdom for their identification in the green alga (Chlamydomonas reinhardtii), the protist (Giardia lamblia) and the slime mold (Dictyostelium discoideum) ^[35]. The WRKY homologues of protist, slime mold and green alga contain two WRKY domains interrupted by an intron. No one of WRKY homologues in moss and ferns resides in group III, while over 20% of the family members in higher plants comprise group III. It seems that members of group III have evolved late and members of group I would be the ancestral type.

As gene orthology would imply similar gene function, the assignment present provides a solid base for comparison of functional analysis between Arabidopsis and rice or other species. Table 2 shows the determined orthology at the functional level from sequence homologies between rice and Arabidopsis or other species. For instance, PcWRKY1 and AtWRKY33 are homologous to OsWRKY53, and these three WRKY proteins referred to defense responses to pathogen infection. PcWRKY1 was elicitor-inducible, overexpression of AtWRKY33 increased resistance to fungal pathogens, and ectopic expression of OsWRKY53 also enhanced resistance to rice blast fungus ^[17]; AtWRKY18 and HvWRKY38 are homologous to OsWRKY71. Both HvWRKY38 and OsWRKY71 were induced by SA and ABA, but suppressed by GA, which suggested that their function refers to defense response and development ^[64], and overexpression lines of AtWRKY18 were the positive regulator of basal defense and SAR, and were stunted in growth, which suggested that the function of AtWRKY18 also referred to plant development ^[82]. Both OsWRKY08 and AtWRKY28 responded to various abiotic stresses and their overexpression Arabidopsis showed the improved tolerance to osmotic stress ^[25].

Clearly, identifying upstream and downstream genes of OsWRKY factors would be crucial in understanding their regulatory net work, and constructing the insertion mutant, artificial micro-RNA, and overexpresion lines of OsWRKY genes should be better to traverse the biological functions of these genes in development, growth, biotic defense, and abiotic tolerance. Currently, the majority of such studies have employed strong ectopic expression of OsWRKY genes in transgenic plants or transient expression aleurone cells except OsWRKY45 knock-down lines, OsWRKY31 RNA interference lines, and OsWRKY13 suppressed plants. So we encourage the researchers on rice WRKY function analysis to respond to the call for an international coordinated effort in rice functional genomics in the form of a project named RICE2020 ^[85].