Caenorhabditis elegans RIG-I-like receptor DRH-1 signals via CARDs to activate antiviral immunity in intestinal cells.

Upon sensing viral RNA, mammalian RIG-I-like receptors (RLRs) activate downstream signals using caspase activation and recruitment domains (CARDs), which ultimately promote transcriptional immune responses that have been well studied. In contrast, the downstream signaling mechanisms for invertebrate RLRs are much less clear. For example, the Caenorhabditis elegans RLR DRH-1 lacks annotated CARDs and up-regulates the distinct output of RNA interference. Here, we found that similar to mammal RLRs, DRH-1 signals through two tandem CARDs (2CARD) to induce a transcriptional immune response. Expression of DRH-1(2CARD) alone in the intestine was sufficient to induce immune gene expression, increase viral resistance, and promote thermotolerance, a phenotype previously associated with immune activation in C. elegans. We also found that DRH-1 is required in the intestine to induce immune gene expression, and we demonstrate subcellular colocalization of DRH-1 puncta with double-stranded RNA inside the cytoplasm of intestinal cells upon viral infection. Altogether, our results reveal mechanistic and spatial insights into antiviral signaling in C. elegans, highlighting unexpected parallels in RLR signaling between C. elegans and mammals.

Microsporidia: Pervasive natural pathogens of Caenorhabditis elegans and related nematodes.

The nematode Caenorhabditis elegans is an invaluable host model for studying infections caused by various pathogens, including microsporidia. Microsporidia represent the first natural pathogens identified in C. elegans, revealing the previously unknown Nematocida genus of microsporidia. Following this discovery, the utilization of nematodes as a model host has rapidly expanded our understanding of microsporidia biology and has provided key insights into the cell and molecular mechanisms of antimicrosporidia defenses. Here, we first review the isolation history, morphological characteristics, life cycles, tissue tropism, genetics, and host immune responses for the four most well-characterized Nematocida species that infect C. elegans. We then highlight additional examples of microsporidia that infect related terrestrial and aquatic nematodes, including parasitic nematodes. To conclude, we assess exciting potential applications of the nematode-microsporidia system while addressing the technical advances necessary to facilitate future growth in this field.

A cullin-RING ubiquitin ligase promotes thermotolerance as part of the intracellular pathogen response in Caenorhabditis elegans.

Intracellular pathogen infection leads to proteotoxic stress in host organisms. Previously we described a physiological program in the nematode Caenorhabditis elegans called the intracellular pathogen response (IPR), which promotes resistance to proteotoxic stress and appears to be distinct from canonical proteostasis pathways. The IPR is controlled by PALS-22 and PALS-25, proteins of unknown biochemical function, which regulate expression of genes induced by natural intracellular pathogens. We previously showed that PALS-22 and PALS-25 regulate the mRNA expression of the predicted ubiquitin ligase component cullin cul-6, which promotes thermotolerance in pals-22 mutants. However, it was unclear whether CUL-6 acted alone, or together with other cullin-ring ubiquitin ligase components, which comprise a greatly expanded gene family in C. elegans Here we use coimmunoprecipitation studies paired with genetic analysis to define the cullin-RING ligase components that act together with CUL-6 to promote thermotolerance. First, we identify a previously uncharacterized RING domain protein in the TRIM family we named RCS-1, which acts as a core component with CUL-6 to promote thermotolerance. Next, we show that the Skp-related proteins SKR-3, SKR-4, and SKR-5 act redundantly to promote thermotolerance with CUL-6. Finally, we screened F-box proteins that coimmunoprecipitate with CUL-6 and find that FBXA-158 and FBXA-75 promote thermotolerance. In summary, we have defined the three core components and two F-box adaptors of a cullin-RING ligase complex that promotes thermotolerance as part of the IPR in C. elegans, which adds to our understanding of how organisms cope with proteotoxic stress.

Functional divergence of GLP genes between G. barbadense and G. hirsutum in response to Verticillium dahliae infection.

Germin-like proteins (GLPs) play important roles in plant disease resistance but are rarely reported in cotton. We compared the expression of GLPs in Verticillium dahliae inoculate G. hirsutum (susceptible) and G. barbadense (resistant) and enriched 11 differentially expressed GLPs. 2741 GLP proteins identified from 53 species determined that GLP probably originated from algae and could be classified into 7 clades according to phylogenetic analysis, among which Clade I is likely the most ancient. Cotton GLP (two allopolyploids and two diploids) genes within a shared clade were highly conserved. Intriguingly, clade VII genes were mainly located in gene clusters that derived from the expansion of LTR transposons. Clade VII members expressed mainly in root which is the first battle against Verticillium dahlia and could be induced more intensely in G. barbadense than G. hirsutum. The GLP genes are resistant to Verticillium dahliae, which can be further investigated against Verticillium wilt.

Enzyme activity profiling for physiological phenotyping within functional phenomics: plant growth and stress responses.

High-throughput profiling of key enzyme activities of carbon, nitrogen, and antioxidant metabolism is emerging as a valuable approach to integrate cell physiological phenotyping into a holistic functional phenomics approach. However, the analyses of the large datasets generated by this method represent a bottleneck, often keeping researchers from exploiting the full potential of their studies. We address these limitations through the exemplary application of a set of data evaluation and visualization tools within a case study. This includes the introduction of multivariate statistical analyses that can easily be implemented in similar studies, allowing researchers to extract more valuable information to identify enzymatic biosignatures. Through a literature meta-analysis, we demonstrate how enzyme activity profiling has already provided functional information on the mechanisms regulating plant development and response mechanisms to abiotic stress and pathogen attack. The high robustness of the distinct enzymatic biosignatures observed during developmental processes and under stress conditions underpins the enormous potential of enzyme activity profiling for future applications in both basic and applied research. Enzyme activity profiling will complement molecular -omics approaches to contribute to the mechanistic understanding required to narrow the genotype-to-phenotype knowledge gap and to identify predictive biomarkers for plant breeding to develop climate-resilient crops.

The ubiquitin system affects agronomic plant traits.

In a single vascular plant species, the ubiquitin system consists of thousands of different proteins involved in attaching ubiquitin to substrates, recognizing or processing ubiquitinated proteins, or constituting or regulating the 26S proteasome. The ubiquitin system affects plant health, reproduction, and responses to the environment, processes that impact important agronomic traits. Here we summarize three agronomic traits influenced by ubiquitination: induction of flowering, seed size, and pathogen responses. Specifically, we review how the ubiquitin system affects expression of genes or abundance of proteins important for determining when a plant flowers (focusing on FLOWERING LOCUS C, FRIGIDA, and CONSTANS), highlight some recent studies on how seed size is affected by the ubiquitin system, and discuss how the ubiquitin system affects proteins involved in pathogen or effector recognition with details of recent studies on FLAGELLIN SENSING 2 and SUPPRESSOR OF NPR CONSTITUTIVE 1, respectively, as examples. Finally, we discuss the effects of pathogen-derived proteins on plant host ubiquitin system proteins. Further understanding of the molecular basis of the above processes could identify possible genes for modification or selection for crop improvement.

Molecular mechanism of modulating miR482b level in tomato with botrytis cinerea infection.

BACKGROUND: Plant miRNAs are involved in the response to biotic and abiotic stresses by altering their expression levels, and they play an important role in the regulation of plant resistance to stress. However, the molecular mechanism that regulates the expression levels of miRNAs in plants with biotic and abiotic stress still needs to be explored. Previously, we found that the expression of the miR482 family was changed in tomato infected by Botrytis cinerea. In this study, we investigated and uncovered the mechanism underlying the response of miR482 to B. cinerea infection in tomato. RESULTS: First, RT-qPCR was employed to detect the expression patterns of miR482b in tomato infected by B. cinerea, and results showed that miR482b primary transcripts (pri-miR482b) were up-regulated in B. cinerea-infected leaves, but the mature miR482b was down-regulated. Subsequently, we used rapid amplification cDNA end method to amplify the full-length of pri-miR482b. Result showed that the pri-miR482b had two isoforms, with the longer one (consisting 300 bp) having an extra fragment of 53 bp in the 3'-end compared with the shorter one. In vitro Dicer assay indicated that the longer isoform pri-miR482b-x1 had higher efficiency in the post-transcriptional splicing of miRNA than the shorter isoform pri-miR482b-x2. In addition, the transcription level of mature miR482b was much higher in transgenic Arabidopsis overexpressing pri-miR482b-x1 than that in OE pri-miR482b-x2 Arabidopsis. These results confirmed that this extra 53 bp in pri-miR482b-x1 might play a key role in the miR482b biogenesis of post-transcription processing. CONCLUSIONS: Extra 53 bp in pri-miR482b-x1 enhanced miR482b biogenesis, which elevated the transcription level of miR482b. This study clarified the response of miR482 to B. cinerea infection in tomato, thereby helping us further understand the molecular mechanisms that regulate the expression levels of other miRNAs.

Stress-Related Changes in the Expression and Activity of Plant Carbonic Anhydrases.

The data on stress-related changes in the expression and activity of plant carbonic anhydrases (CAs) suggest that they are generally upregulated at moderate stress severity. This indicates probable involvement of CAs in adaptation to drought, high salinity, heat, high light, Ci deficit, and excess bicarbonate. The changes in CA levels under cold stress are less studied and generally represented by the downregulation of CAs excepting ßCA2. Excess Cd2+ and deficit of Zn2+ specifically reduce CA activity and reduce its synthesis. Probable roles of ßCAs in stress adaptation include stomatal closure, ROS scavenging and partial compensation for decreased mesophyll CO2 conductance. ßCAs play contrasting roles in pathogen responses, interacting with phytohormone signaling networks. Their role can be either negative or positive, probably depending on the host-pathogen system, pathogen initial titer, and levels of ·NO and ROS. It is still not clear why CAs are suppressed under severe stress levels. It should be noted, that the role of ßCAs in the facilitation of CO2 diffusion and their involvement in redox signaling or ROS detoxication are potentially antagonistic, as they are inactivated by oxidation or nitrosylation. Interestingly, some chloroplastic ßCAs may be relocated to the cytoplasm under stress conditions, but the physiological meaning of this effect remains to be studied.

The BTB/POZ domain protein GmBTB/POZ promotes the ubiquitination and degradation of the soybean AP2/ERF-like transcription factor GmAP2 to regulate the defense response to Phytophthora sojae.

Phytophthora root and stem rot in soybean (Glycine max) is a destructive disease worldwide, and hence improving crop resistance to the causal pathogen, P. sojae, is a major target for breeders. However, it remains largely unclear how the pathogen regulates the various affected signaling pathways in the host, which consist of complex networks including key transcription factors and their targets. We have previously demonstrated that GmBTB/POZ enhances soybean resistance to P. sojae and the associated defense response. Here, we demonstrate that GmBTB/POZ interacts with the transcription factor GmAP2 and promotes its ubiquitination. GmAP2-RNAi transgenic soybean hairy roots exhibited enhanced resistance to P. sojae, whereas roots overexpressing GmAP2 showed hypersensitivity. GmWRKY33 was identified as a target of GmAP2, which represses its expression by directly binding to the promoter. GmWRKY33 acts as a positive regulator in the response of soybean to P. sojae. Overexpression of GmBTB/POZ released the GmAP2-regulated suppression of GmWRKY33 in hairy roots overexpressing GmAP2 and increased their resistance to P. sojae. Taken together, our results indicate that GmBTB/POZ-GmAP2 modulation of the P. sojae resistance response forms a novel regulatory mechanism, which putatively regulates the downstream target gene GmWRKY33 in soybean.

Suppression of NB-LRR genes by miRNAs promotes nitrogen-fixing nodule development in Medicago truncatula.

Plant genomes contain two major classes of innate immune receptors to recognize different pathogens. The pattern recognition receptors perceive conserved pathogen-associated molecular patterns and the resistance genes with nucleotide-binding (NB) and leucine-rich repeat (LRR) domains recognize specific pathogen effectors. The precise regulation of resistance genes is important since the unregulated expression of NB-LRR genes can inhibit growth and may result in autoimmunity in the absence of pathogen infection. It was shown that a subset of miRNAs could target NB-LRR genes and act as an important regulator of plant immunity in the absence of pathogens. Plants not only interact with pathogens, but they can also establish symbiotic interactions with microbes. Nitrogen-fixing symbiotic interaction and nodule formation of legumes may also require the suppression of host defence to prevent immune responses. We found that upon symbiotic interactions, miRNAs repressing NB-LRR expression are upregulated in the developing nodules of Medicago truncatula. Furthermore, we show that the suppression of the activity of the NB-LRR genes targeted by these miRNAs is important during nodule development. Our results suggest that the downregulation of NB-LRR resistance genes in the developing nodule produces a suitable niche that facilitates bacterial colonization and the development of an N-fixing nodule.

Current concepts and an alternative perspective on periodontal disease.

BACKGROUND: Epidemiological data from countries worldwide show a consistent pattern implying that a fraction of around 10% of those over 40-50 years in all populations will exhibit severe periodontitis with the potential risk of losing teeth during their life-time. The subgingival microbiota shows striking similarities between populations irrespective of disease severity and can only marginally explain the clinical pattern. It is also difficult to explain this pattern by genetic and acquired risk factors such as systemic disease (e.g. diabetes) or habits (e.g. smoking) even if they may have a confounding effect on the disease. MAIN TEXT: Inflammation of the gingiva appears to be a normal and physiological response to the presence of commensal bacteria along the gingival crevice and in the dental biofilm. Over many years of exposure to the dental biofilm, the chronic inflammation in the gingiva gradually results in a loss of attachment and bone loss. Numerous laboratory and clinical studies have provided insight into the potential role of determinants that are associated with periodontitis. However, it has been difficult to relate the findings to the pattern of the distribution of the disease observed in epidemiological studies. We propose a simple and parsimonious model that considers all the multitude of potential determinants as creating effectively random noise within the dental biofilm to which the tissues react by accumulating the effects of this noise. CONCLUSIONS: We suggest that such a model can explain many of the epidemiological features of periodontal breakdown over time, and we discuss its clinical implications.

The ancient cytokine IL-17D is regulated by Nrf2 and mediates tumor and virus surveillance.

Early stage immune responses can dictate the severity and outcome of inflammatory processes such as tumor growth and viral infection. Cytokines such as the interleukin 17 (IL-17) family and cellular stress defense (e.g., anti-oxidant) pathways have evolved early and regulate disease surveillance in vertebrates and invertebrates as far back as Caenorhabditis elegans. Our group has recently found a new role for nuclear factor erythroid-derived 2-like 2 (Nrf2) in regulating early anti-cancer immune responses by inducing IL-17D and recruiting natural killer (NK) cells. In this Cytokine Stimulus, we discuss recent findings that encourage boosting the Nrf2/IL-17D/NK cell axis for the treatment of cancer and viral infection.

Host responses and bacterial colonization following inoculation of Pasteurella multocida P52 strain into unvaccinated and aluminum hydroxide gel hemorrhagic septicemia vaccinated mice.

Hemorrhagic septicemia is a highly infectious and contagious disease caused by Pasteurella multocida serogroup B:2 in tropical Asian and African countries. The acute inflammatory responses induced by Pasteurella multocida are the main cause of death in hemorrhagic septicemia. Therefore, present study was undertaken to examine the blood cytokine expression profiles (TNF-α, IL-1ß, and IL-6), bacterial colonization and histopathological changes of intraperitoneally and subcutaneously challenged vaccinated and unvaccinated mice with 102 CFU of P. multocida P52. The observations were made at 6, 12, 18, 24 h and 48 h intervals. Real-time PCR based blood cytokine profiles (TNF-α, IL-1ß, and IL-6) measurement revealed a significantly higher amount of pro-inflammatory cytokines expression in the unvaccinated challenged group of mice than the vaccinated challenged group. There was heavy bacterial load in all organs of mice viz. trachea, lung, spleen, within 6 h of challenge in both vaccinated and unvaccinated group of mice, but bacterial load increased in the unvaccinated challenged group of mice with respect to time whereas the load were constant in the vaccinated challenged group. Histopathological changes were mild in the vaccinated challenged group of mice in comparison to the unvaccinated challenged group. There was no significant difference in the bacterial load, histopathological changes and cytokines expression when challenged through different routes.

In silico analysis of the grapefruit sRNAome, transcriptome and gene regulation in response to CTV-CDVd co-infection.

BACKGROUND: Small RNA (sRNA) associated gene regulation has been shown to play a significant role during plant-pathogen interaction. In commercial citrus orchards co-infection of Citrus tristeza virus (CTV) and viroids occur naturally. METHODS: A next-generation sequencing-based approach was used to study the sRNA and transcriptional response in grapefruit to the co-infection of CTV and Citrus dwarfing viroid. RESULTS: The co-infection resulted in a difference in the expression of a number of sRNA species when comparing healthy and infected plants; the majority of these were derived from transcripts processed in a phased manner. Several RNA transcripts were also differentially expressed, including transcripts derived from two genes, predicted to be under the regulation of sRNAs. These genes are involved in plant hormone systems; one in the abscisic acid, and the other in the cytokinin regulatory pathway. Additional analysis of virus- and viroid-derived small-interfering RNAs (siRNAs) showed areas on the pathogen genomes associated with increased siRNA synthesis. Most interestingly, the starting position of the p23 silencing suppressor's sub-genomic RNA generated a siRNA hotspot on the CTV genome. CONCLUSIONS: This study showed the involvement of various genes, as well as endogenous and exogenous RNA-derived sRNA species in the plant-defence response. The results highlighted the role of sRNA-directed plant hormone regulation during biotic stress, as well as a counter-response of plants to virus suppressors of RNA-silencing.

Plant resistance against the parasitic nematode Heterodera schachtii is mediated by MPK3 and MPK6 kinases, which are controlled by the MAPK phosphatase AP2C1 in Arabidopsis.

Plant-parasitic cyst nematodes infect plants and form highly sophisticated feeding sites in roots. It is not known which plant cell signalling mechanisms trigger plant defence during the early stages of nematode parasitism. Mitogen-activated protein kinases (MAPKs) are central components of protein phosphorylation cascades transducing extracellular signals to plant defence responses. MAPK phosphatases control kinase activities and the signalling outcome. The involvement and the role of MPK3 and MPK6, as well as the MAPK phosphatase AP2C1, is demonstrated during parasitism of the beet cyst nematode Heterodera schachtii in Arabidopsis. Our data reveal notable activation patterns of plant MAPKs and the induction of AP2C1 suggesting the attenuation of defence signalling in plant cells during early nematode infection. It is demonstrated that the ap2c1 mutant that is lacking AP2C1 is more attractive but less susceptible to nematodes compared with the AP2C1-overexpressing line. This implies that the function of AP2C1 is a negative regulator of nematode-induced defence. By contrast, the enhanced susceptibility of mpk3 and mpk6 plants indicates a positive role of stress-activated MAPKs in plant immunity against nematodes. Evidence is provided that phosphatase AP2C1, as well as AP2C1-targeted MPK3 and MPK6, are important regulators of plant-nematode interaction, where the co-ordinated action of these signalling components ensures the timely activation of plant defence.

The zinc-binding nuclear protein HIPP3 acts as an upstream regulator of the salicylate-dependent plant immunity pathway and of flowering time in Arabidopsis thaliana.

Biotic and abiotic stress responses of plants are linked to developmental programs. Proteins involved in different signaling pathways are the molecular basis of this concerted interplay. In our study, we show that Arabidopsis thaliana HEAVY METAL-ASSOCIATED ISOPRENYLATED PLANT PROTEIN3 (HIPP3; At5g60800) acts as an upstream regulator of stress- and development-related regulatory networks. Localization, metal-binding and stress-responsive gene expression of HIPP3 were analyzed via microscopy, protein and inductively coupled plasma (ICP)-MS analyses and quantitative real-time PCR. In addition, transcriptome and phenotype analyses of plants overexpressing HIPP3 were used to unravel its function. Our data show that HIPP3 is a nuclear, zinc-binding protein. It is repressed during drought stress and abscisic acid (ABA) treatment and, similar to other pathogen-related genes, is induced after infection with Pseudomonas syringae pv. tomato. HIPP3 overexpression affects the regulation of > 400 genes. Strikingly, most of these genes are involved in pathogen response, especially in the salicylate pathway. In addition, many genes of abiotic stress responses and seed and flower development are affected by HIPP3 overexpression. Plants overexpressing HIPP3 show delayed flowering. We conclude that HIPP3 acts via its bound zinc as an upstream regulator of the salicylate-dependent pathway of pathogen response and is also involved in abiotic stress responses and seed and flower development.

Ascorbate deficiency influences the leaf cell wall glycoproteome in Arabidopsis thaliana.

The cell wall forms the first line of interaction between the plant and the external environment. Based on the observation that ascorbate-deficient vtc mutants of Arabidopsis thaliana have increased cell wall peroxidase activity, the cell wall glycoproteome of vtc2-2 was investigated. Glycoproteins were purified from fully expanded leaves by Concanavalin A affinity chromatography and analysed by liquid chromatography quadrupole time-of-flight mass spectrometry. This procedure identified 63 proteins with predicted glycosylation sites and cell wall localization. Of these, 11 proteins were differentially expressed between vtc2-2 and wild type. In particular, PRX33/34 were identified as contributing to increased peroxidase activity in response to ascorbate deficiency. This is the same peroxidase previously shown to contribute to hydrogen peroxide generation and pathogen resistance. Three fasciclin-like arabinogalactan proteins (FLA1, 2 and 8) had lower abundance in vtc2-2. Inspection of published microarray data shows that these also have lower gene expression in vtc1 and vtc2-1 and are decreased in expression by pathogen challenge and oxidative stresses. Ascorbate deficiency therefore impacts expression of cell wall proteins involved in pathogen responses and these presumably contribute to the increased resistance of vtc mutants to biotrophic pathogens.

Robust anti-oxidant defences in the rice blast fungus Magnaporthe oryzae confer tolerance to the host oxidative burst.

Plants respond to pathogen attack via a rapid burst of reactive oxygen species (ROS). However, ROS are also produced by fungal metabolism and are required for the development of infection structures in Magnaporthe oryzae. To obtain a better understanding of redox regulation in M. oryzae, we measured the amount and redox potential of glutathione (E(GSH)), as the major cytoplasmic anti-oxidant, the rates of ROS production, and mitochondrial activity using multi-channel four-dimensional (x,y,z,t) confocal imaging of Grx1-roGFP2 and fluorescent reporters during spore germination, appressorium formation and infection. High levels of mitochondrial activity and ROS were localized to the growing germ tube and appressorium, but E(GSH) was highly reduced and tightly regulated during development. Furthermore, germlings were extremely resistant to external H2O2 exposure ex planta. EGSH remained highly reduced during successful infection of the susceptible rice cultivar CO39. By contrast, there was a dramatic reduction in the infection of resistant (IR68) rice, but the sparse hyphae that did form also maintained a similar reduced E(GSH). We conclude that M. oryzae has a robust anti-oxidant defence system and maintains tight control of EGSH despite substantial oxidative challenge. Furthermore, the magnitude of the host oxidative burst alone does not stress the pathogen sufficiently to prevent infection in this pathosystem.

Callose biosynthesis in Arabidopsis with a focus on pathogen response: what we have learned within the last decade.

BACKGROUND: (1,3)-ß-Glucan callose is a cell wall polymer that is involved in several fundamental biological processes, ranging from plant development to the response to abiotic and biotic stresses. Despite its importance in maintaining plant integrity and plant defence, knowledge about the regulation of callose biosynthesis at its diverse sites of action within the plant is still limited. The moderately sized family of GSL (GLUCAN SYNTHASE-LIKE) genes is predicted to encode callose synthases with a specific biological function and subcellular localization. Phosphorylation and directed translocation of callose synthases seem to be key post-translational mechanisms of enzymatic regulation, whereas transcriptional control of GSL genes might only have a minor function in response to biotic or abiotic stresses. SCOPE AND CONCLUSIONS: Among the different sites of callose biosynthesis within the plant, particular attention has been focused on the formation of callose in response to pathogen attack. Here, callose is deposited between the plasma membrane and the cell wall to act as a physical barrier to stop or slow invading pathogens. Arabidopsis (Arabidopsis thaliana) is one of the best-studied models not only for general plant defence responses but also for the regulation of pathogen-induced callose biosynthesis. Callose synthase GSL5 (GLUCAN SYNTHASE-LIKE5) has been shown to be responsible for stress-induced callose deposition. Within the last decade of research into stress-induced callose, growing evidence has been found that the timing of callose deposition in the multilayered system of plant defence responses could be the key parameter for optimal effectiveness. This timing seems to be achieved through co-ordinated transport and formation of the callose synthase complex.

Multiple functions and regulatory networks of WRKY33 and its orthologs.

Arabidopsis thaliana WRKY33 is currently one of the most studied members of the Group I WRKY transcription factor family. Research has confirmed that WRKY33 is involved in the regulation of various biological and abiotic stresses and occupies a central position in the regulatory network. The functional studies of orthologous genes of WRKY33 from other species are also receiving increasing attention. In this article, we summarized thirty-eight orthologous genes of AtWKRY33 from twenty-five different species. Their phylogenetic relationship and conserved WRKY domain were analyzed and compared. Similar to AtWKRY33, the well-studied orthologous gene members from rice and tomato also have multiple functions. In addition to playing important regulatory roles in responding to their specific pathogens, they are also involved in regulating various abiotic stresses and development. AtWKRY33 exerts its multiple functions through a complex regulatory network. Upstream transcription factors or other regulatory factors activate or inhibit the expression of AtWKRY33 at the chromatin and transcriptional levels. Interacting proteins affect the transcriptional activity of AtWKRY33 through phosphorylation, ubiquitination, SUMOylation, competition, or cooperation. The downstream genes are diverse and include three major categories: transcription factors, synthesis, metabolism, and signal transduction of various hormones, and disease resistance genes. In the regulatory network of AtWRKY33 orthologs, many conserved regulatory characteristics have been discovered, such as self-activation and phosphorylation by MAP kinases. This can provide a comparative reference for further studying the functions of other orthologous genes of AtWKRY33.