Guoyin Liu

RAV transcription factors are essential for disease resistance against cassava bacterial blight via activation of melatonin biosynthesis genes

With 1 AP2 domain and 1 B3 domain, 7 MeRAVs in apetala2/ethylene response factor (AP2/ERF) gene family have been identified in cassava. However, the in vivo roles of these remain unknown. Gene expression assays showed that the transcripts of MeRAVs were commonly regulated after Xanthomonas axonopodis pv manihotis (Xam) and MeRAVs were specifically located in plant cell nuclei. Through virus‐induced gene silencing (VIGS) in cassava, we found that MeRAV1 and MeRAV2 are essential for plant disease resistance against cassava bacterial blight, as shown by the bacterial propagation of Xam in plant leaves. Through VIGS in cassava leaves and overexpression in cassava leave protoplasts, we found that MeRAV1 and MeRAV2 positively regulated melatonin biosynthesis genes and the endogenous melatonin level. Further investigation showed that MeRAV1 and MeRAV2 are direct transcriptional activators of 3 melatonin biosynthesis genes in cassava, as evidenced by chromatin immunoprecipitation‐PCR in cassava leaf protoplasts and electrophoretic mobility shift assay. Moreover, cassava melatonin biosynthesis genes also positively regulated plant disease resistance. Taken together, this study identified MeRAV1 and MeRAV2 as common and upstream transcription factors of melatonin synthesis genes in cassava and revealed a model of MeRAV1 and MeRAV2‐melatonin biosynthesis genes‐melatonin level in plant disease resistance against cassava bacterial blight.

Two transcriptional activators of N-acetylserotonin O-methyltransferase 2 and melatonin biosynthesis in cassava

Similar to the situation in animals, melatonin biosynthesis is regulated by four sequential enzymatic steps in plants. Although the melatonin synthesis genes have been identified in various plants, the upstream transcription factors of them remain unknown. In this study on cassava (Manihot esculenta), we found that MeWRKY79 and heat-shock transcription factor 20 (MeHsf20) targeted the W-box and the heat-stress elements (HSEs) in the promoter of N-acetylserotonin O-methyltransferase 2 (MeASMT2), respectively. The interaction between MeWRKY79, MeHsf20, and the MeASMT2 promoter was evidenced by the activation of promoter activity and chromatin immunoprecipitation (ChIP) in cassava protoplasts, and by an in vitro electrophoretic mobility shift assay (EMSA). The transcripts of MeWRKY79, MeHsf20, and MeASMT2 were all regulated by a 22-amino acid flagellin peptide (flg22) and by Xanthomonas axonopodis pv manihotis (Xam). In common with the phenotype of MeASMT2, transient expression of MeWRKY79 and MeHsf20 in Nicotiana benthamiana leaves conferred improved disease resistance. Through virus-induced gene silencing (VIGS) in cassava, we found that MeWRKY79- and MeHsf20-silenced plants showed lower transcripts of MeASMT2 and less accumulation of melatonin, which resulted in disease sensitivity that could be reversed by exogenous melatonin. Taken together, these results indicate that MeASMT2 is a target of MeWRKY79 and MeHsf20 in plant disease resistance. This study identifies novel upstream transcription factors of melatonin synthesis genes in cassava, thus extending our knowledge of the complex modulation of melatonin synthesis in plant defense.

Melatonin biosynthesis enzymes recruit WRKY transcription factors to regulate melatonin accumulation and transcriptional activity on W-box in cassava

Melatonin is widely involved in growth, development, and stress responses in plants. Although the melatonin synthesis enzymes have been identified in various plants, their interacting proteins remain unknown. Herein, overexpression of tryptophan decarboxylase 2 (MeTDC2)‐interacting proteins, N‐acetylserotonin O‐methyltransferase 2 (MeASMT2) interacting proteins, and N‐acetylserotonin O‐methyltransferase 3 (MeASMT3) in cassava leaf protoplasts resulted in more melatonin than when other enzymes were overexpressed. Through yeast two‐hybrid, 14 MeTDC2‐interacting proteins, 24 MeASMT2 interacting proteins, and 9 MeASMT3‐interacting proteins were identified. Notably, we highlighted MeWRKY20 and MeWRKY75 as common interacting proteins of the 3 enzymes, as evidenced by yeast two‐hybrid, and in vivo bimolecular fluorescence complementation (BiFC). Moreover, co‐overexpression of MeTDC2/MeASMT2/3 with MeWRKY20/75 in cassava leaf protoplasts did not only activated the transcriptional activities of MeWRKY20 and MeWRKY75 on W‐box, but also induced the effects of MeTDC2, MeASMT2/3 on endogenous melatonin levels. Taken together, 3 melatonin synthesis enzymes (MeTDC2, MeASMT2/3) interact with MeWRKY20/75 to form a protein complex in cassava. This information significantly extends the knowledge of the complex modulation of plant melatonin signaling.

Identification and functional analysis of cassava DELLA proteins in plant disease resistance against cassava bacterial blight

Gibberellin (GA) is an essential plant hormone in plant growth and development as well as various stress responses. DELLA proteins are important repressors of GA signal pathway. GA and DELLA have been extensively investigated in several model plants. However, the in vivo roles of GA and DELLA in cassava, one of the most important crops and energy crops in the tropical area, are unknown. In this study, systematic genome-wide analysis identified 4 MeDELLAs in cassava, as evidenced by the evolutionary tree, gene structures and motifs analyses. Gene expression analysis found that 4 MeDELLAs were commonly regulated by flg22 and Xanthomonas axonopodis pv manihotis (Xam). Through overexpression in Nicotiana benthamiana, we found that 4 MeDELLAs conferred improved disease resistance against cassava bacterial blight. Through virus-induced gene silencing (VIGS) in cassava, we found that MeDELLA-silenced plants exhibited decreased disease resistance, with less callose deposition and lower transcript levels of defense-related genes. This is the first study identifying MeDELLAs as positive regulators of disease resistance against cassava bacterial blight.

Genome-wide analysis of autophagy-related genes in banana highlights MaATG8s in cell death and autophagy in immune response to Fusarium wilt

Key messageMaATG8s play important roles in hypersensitive-like cell death and immune response, and autophagy is essential for disease resistance againstFoc in banana.AbstractAutophagy is responsible for the degradation of damaged cytoplasmic constituents in the lysosomes or vacuoles. Although the effects of autophagy have been extensively revealed in model plants, the possible roles of autophagy-related gene in banana remain unknown. In this study, 32 MaATGs were identified in the draft genome, and the profiles of several MaATGs in response to fungal pathogen Fusarium oxysporum f. sp. cubense (Foc) were also revealled. We found that seven MaATG8s were commonly regulated by Foc. Through transient expression in Nicotiana benthamiana leaves, we highlight the novel roles of MaATG8s in conferring hypersensitive-like cell death, and MaATG8s-mediated hypersensitive response-like cell death is dependent on autophagy. Notablly, autophagy inhibitor 3-methyladenine (3-MA) treatment resulted in decreased disease resistance in response to Foc4, and the effect of 3-MA treatment could be rescued by exogenous salicylic acid, jasmonic acid and ethylene, indicating the involvement of autophagy-mediated plant hormones in banana resistance to Fusarium wilt. Taken together, this study may extend our understanding the putative role of MaATG8s in hypersensitive-like cell death and the essential role of autophagy in immune response against Foc in banana.

Two Cassava Basic Leucine Zipper (bZIP) Transcription Factors (MebZIP3 and MebZIP5) Confer Disease Resistance against Cassava Bacterial Blight

Basic domain-leucine zipper (bZIP) transcription factor, one type of conserved gene family, plays an important role in plant development and stress responses. Although 77 MebZIPs have been genome-wide identified in cassava, their in vivo roles remain unknown. In this study, we analyzed the expression pattern and the function of two MebZIPs (MebZIP3 and MebZIP5) in response to pathogen infection. Gene expression analysis indicated that MebZIP3 and MebZIP5 were commonly regulated by flg22, Xanthomonas axonopodis pv. manihotis (Xam), salicylic acid (SA), and hydrogen peroxide (H2O2). Subcellular localization analysis showed that MebZIP3 and MebZIP5 are specifically located in cell nucleus. Through overexpression in tobacco, we found that MebZIP3 and MebZIP5 conferred improved disease resistance against cassava bacterial blight, with more callose depositions. On the contrary, MebZIP3- and MebZIP5-silenced plants by virus-induced gene silencing (VIGS) showed disease sensitive phenotype, lower transcript levels of defense-related genes and less callose depositions. Taken together, this study highlights the positive role of MebZIP3 and MebZIP5 in disease resistance against cassava bacterial blight for further utilization in genetic improvement of cassava disease resistance.

Zinc finger of Arabidopsis thaliana 6 is involved in melatonin-mediated auxin signaling through interacting INDETERMINATE DOMAIN15 and INDOLE-3-ACETIC ACID 17

Although accumulating evidence demonstrates the cross talk between melatonin and auxin as derivatives of tryptophan, the underlying signaling events remain unclear. In this study, we found that melatonin and auxin mediated the transcriptional levels of zinc finger of Arabidopsis thaliana (ZAT6) in a mutually antagonistic manner. ZAT6 negatively modulated the endogenous auxin level, and ZAT6 knockdown plants were less sensitive to melatonin‐regulated auxin biosynthesis, indicating its involvement in melatonin‐mediated auxin accumulation. Additionally, the identification of INDETERMINATE DOMAIN15 (IDD15) and INDOLE‐3‐ACETIC ACID 17 (IAA17) in Arabidopsis that interacted with ZAT6 in vivo provided new insight of ZAT6‐mediated auxin signaling. Further investigation showed that ZAT6 repressed the transcription activation of IDD15 on the YUC2 promoter, while ZAT6 inhibited the interaction of TRANSPORT INHIBITOR RESPONSE 1 (TIR1) and IAA17 through competitively binding to IAA17. Thus, both auxin synthesis and the auxin response were negatively modulated by ZAT6. Taken together, ZAT6 is involved in melatonin‐mediated auxin signaling through forming an interacting complex of auxin signaling pathway in Arabidopsis.

Functional characterization of WHY–WRKY75 transcriptional module in plant response to cassava bacterial blight

Cassava is a major food crop in tropical areas, but its productivity and quality are seriously limited by cassava bacterial blight. So far, the key factors regulating cassava immune response remain elusive. In this study, we identified three cassava Whirly genes (MeWHYs) in cassava variety of South China 124 (SC124), and explored the possible roles and utilization of MeWHYs in cassava disease resistance. Gene expression analysis revealed that the transcripts of three MeWHYs were commonly regulated by the highly conserved N-terminal epitope of f lagellin (flg22) and Xanthomonas axonopodis pv. manihotis Hainan (Xam HN) treatments. Overexpression of MeWHYs improved plant disease resistance against X. axonopodis pv. manihotis, while MeWHYs-silenced cassava plants by virus-induced gene silencing exhibited decreased disease resistance. Notably, MeWRKY75 physically interacted with three MeWHYs in yeast and in planta, and served as a transcriptional activator of MeWHY3. Moreover, the physical interaction between MeWHYs and MeWRKY75 promoted the transcriptional activities of each other. Consistently, MeWRKY75 also positively regulated disease resistance against cassava bacterial blight. Taken together, our observations suggested that MeWRKY75 and MeWHYs confer improved disease resistance against cassava bacterial blight through forming an interacting complex of MeWRKY75-MeWHY1/2/3 and transcriptional module of MeWRKY75-MeWHY3. This study facilitates our understanding of the positive effect of the MeWRKY75-MeWHY3 transcriptional module in plant disease resistance.

The zinc-finger transcription factor ZAT6 is essential for hydrogen peroxide induction of anthocyanin synthesis in Arabidopsis

The accumulation of flavonoids is activated by various abiotic stresses, and the induction of reactive oxygen species (ROS) especially hydrogen peroxide (H2O2) is a general response to abiotic stress in plants. However, the direct link between flavonoids and H2O2 and underlying mechanism remain elusive. In this study, we found that the concentrations of anthocyanin and flavonoids were significantly induced by H2O2 treatment. Furthermore, we found that the transcript level of ZINC FINGER of ARABIDOPSIS THALIANA 6 (ZAT6) was significantly activated after exogenous H2O2 treatment, and modulation of AtZAT6 expression positively affected the concentrations of both anthocyanin and total flavonoids. Notably, exogenous H2O2-induced anthocyanin synthesis was largely alleviated in AtZAT6 knockdown plants, but showed higher level in AtZAT6 overexpressing plants. AtZAT6 directly activated the expressions of TT5, TT7, TT3, TT18, MYB12, and MYB111 through binding to their promoters with TACAAT elements of these genes, and the activation of MYB12 and MYB111 up-regulated the expressions of TT4 and TT6. Taken together, this study indicates that AtZAT6 plays important role in H2O2-activated anthocyanin synthesis, via directly binding to the promoters of several genes that involved in anthocyanin synthesis.

Molecular identification of GAPDHs in cassava highlights the antagonism of MeGAPCs and MeATG8s in plant disease resistance against cassava bacterial blight

AbstractKey messageMeGAPCs were identified as negative regulators of plant disease resistance, and the interaction of MeGAPCs and MeATG8s was highlighted in plant defense response.AbstractAs an important enzyme of glycolysis metabolic pathway, glyceraldehyde-3-P dehydrogenase (GAPDH) plays important roles in plant development, abiotic stress and immune responses. Cassava (Manihot esculenta) is most important tropical crop and one of the major food crops, however, no information is available about GAPDH gene family in cassava. In this study, 14 MeGAPDHs including 6 cytosol GAPDHs (MeGAPCs) were identified from cassava, and the transcripts of 14 MeGAPDHs in response to Xanthomonas axonopodis pv manihotis (Xam) indicated their possible involvement in immune responses. Further investigation showed that MeGAPCs are negative regulators of disease resistance against Xam. Through transient expression in Nicotiana benthamiana, we found that overexpression of MeGAPCs led to decreased disease resistance against Xam. On the contrary, MeGAPCs-silenced cassava plants through virus-induced gene silencing (VIGS) conferred improved disease resistance. Notably, MeGAPCs physically interacted with autophagy-related protein 8b (MeATG8b) and MeATG8e and inhibited autophagic activity. Moreover, MeATG8b and MeATG8e negatively regulated the activities of NAD-dependent MeGAPDHs, and are involved in MeGAPCs-mediated disease resistance. Taken together, this study highlights the involvement of MeGAPCs in plant disease resistance, through interacting with MeATG8b and MeATG8e.