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. 2018 Jul 19;19(10):2209–2220. doi: 10.1111/mpp.12691

Heat shock transcription factor 3 regulates plant immune response through modulation of salicylic acid accumulation and signalling in cassava

Yunxie Wei 1, Guoyin Liu 1, Yanli Chang 1, Chaozu He 1, Haitao Shi 1,✉
PMCID: PMC6638013  PMID: 29660238

Summary

As the terminal components of signal transduction, heat stress transcription factors (Hsfs) mediate the activation of multiple genes responsive to various stresses. However, the information and functional analysis are very limited in non‐model plants, especially in cassava (Manihot esculenta), one of the most important crops in tropical areas. In this study, 32 MeHsfs were identified from the cassava genome; the evolutionary tree, gene structures and motifs were also analysed. Gene expression analysis found that MeHsfs were commonly regulated by Xanthomonas axonopodis pv. manihotis (Xam). Amongst these MeHsfs, MeHsf3 was specifically located in the cell nucleus and showed transcriptionally activated activity on heat stress elements (HSEs). Through transient expression in Nicotiana benthamiana leaves and virus‐induced gene silencing (VIGS) in cassava, we identified the essential role of MeHsf3 in plant disease resistance, by regulating the transcripts of Enhanced Disease Susceptibility 1 (EDS1) and pathogen‐related gene 4 (PR4). Notably, as regulators of defence susceptibility, MeEDS1 and MePR4 were identified as direct targets of MeHsf3. Moreover, the disease sensitivity of MeHsf3‐ and MeEDS1‐silenced plants could be restored by exogenous salicylic acid (SA) treatment. Taken together, this study highlights the involvement of MeHsf3 in defence resistance through the transcriptional activation of MeEDS1 and MePR4.

Keywords: cassava, defence resistance, heat shock transcription factor (Hsf), pathogen‐related protein, salicylic acid (SA), Xanthomonas axonopodis pv. manihotis (Xam)

Introduction

Under various stress conditions, plants can trigger protective responses through cell membrane sensors, second messengers, kinases, transcription factors and underlying activation of defence‐related genes (Kotak et al., 2004; Singh et al., 2012; Stief et al., 2014; Tanabe et al., 2015). As the terminal components of signal transduction, heat stress transcription factors (Hsfs) mediate the activation of multiple genes responsive to various stresses, including heat stress and other stresses (Almoguera et al., 2015; Charng et al., 2007; Cheng et al., 2015; Giesguth et al., 2015; Hwang et al., 2014; Nishizawa et al., 2006; Nishizawa‐Yokoi et al., 2011; Shim et al., 2009; Yamada and Nishimura, 2008). Hsfs are a class of evolutionarily conserved transcription factors and can be classified into three types: classes A (A1–A9), B (B1–B4) and C (C1 and C2). Hsfs usually contain several conserved domains: (i) the N‐terminal DNA‐binding domain (DBD), which is responsible for the recognition of heat stress elements (HSEs, 5′‐GAAnnTTC‐3′) in promoters of several heat stress‐inducible genes; (ii) the peculiarities of the bipartite oligomerization domain (HR‐A/B); and (iii) other domains including the nuclear export signal (NES), nuclear localization signal (NLS) and C‐terminal activator AHA motif (Huang et al., 2015; Singh et al., 2012; Yabuta, 2016).

Hsfs were initially characterized and cloned in yeast (Saccharomyces cerevisiae) (Sorger and Pelham, 1988), followed by various plants (Huang et al., 2015; Singh et al., 2012; Yabuta, 2016). With the genome sequencing of more and more plant species, the Hsf gene family has been thoroughly identified and characterized in alfalfa (Friedberg et al., 2006), Arabidopsis thaliana (Guo et al., 2008), rice (Oryza sativa L.) (Chauhan et al., 2011; Jin et al., 2013; Wang et al., 2009), maize (Lin et al., 2011), Populus trichocarpa and Medicago truncatula (Wang et al., 2012), Glycine max (Chung et al., 2013), wheat (Chauhan et al., 2013), Chinese cabbage (Song et al., 2014), pepper (Capsicum annuum L.) (Guo et al., 2014, 2015), legume (Lin et al., 2014), cotton (Gossypium hirsutum) (Wang et al., 2014), soybean (Li et al., 2014), pear (Pyrus bretschneideri) (Qiao et al., 2015), Brassica rapa pekinensis (Huang et al., 2015), Populus euphratica (Shen et al., 2015), tea plant (Camellia sinensis) (Liu et al., 2016), strawberry (Fragaria vesca) (Hu Y et al., 2015) and wild Chinese grapevine (Vitis pseudoreticulata) (Hu et al., 2016). So far, plant Hsfs have been found to be widely involved in plant development, heat stress, drought stress, salt stress and the plant disease response (Almoguera et al., 2015; Cheng et al., 2015; Giesguth et al., 2015; Hwang et al., 2014; Tanabe et al., 2015; Yabuta, 2016). In Arabidopsis, overexpressing lines of HSFA1b show greater seed yield and water productivity, and tolerance to drought and biotic stress (Pseudomonas syringae pv. tomato DC3000 and Hyaloperonospora arabidopsidis strain WAC09), than wild‐type plants (Bechtold et al., 2013). AtHsfA6a, markedly induced by exogenous abscisic acid (ABA), NaCl and drought, positively regulates salt and drought stresses by the ABA‐dependent signalling pathway (Hwang et al., 2014). AtHSFA8 is involved in redox translocation from the cytosol to the nucleus (Giesguth et al., 2015). Chen et al. (2015) confirmed that OsHSFA2dI participates in the unfolded protein response via the activation of the expression of unfolded protein response sensors. Previously, research has paid most attention to Hsfs in Arabidopsis and rice (Almoguera et al., 2015; Charng et al., 2007; Cheng et al., 2015; Giesguth et al., 2015; Hwang et al., 2014; Nishizawa et al., 2006; Nishizawa‐Yokoi et al., 2011; Shim et al., 2009; Singh et al., 2012; Tanabe et al., 2015; Yamada and Nishimura, 2008). Information and functional analysis are very limited in non‐model plants, especially in tropical crops.

Cassava (Manihot esculenta) is one of the most important staple crops in tropical areas (Okogbenin et al., 2013; Wei et al., 2016). It is particularly tolerant to drought and low‐fertility soils, and is highly efficient in photosynthesis and starch production; however, it is susceptible to bacterial blight, such as Xanthomonas axonopodis pv. manihotis (Xam) (Boher et al., 1995; Wydra et al., 2007). Moreover, the molecular mechanism of the immune response of cassava and the functional identification of pathogen‐related protein in cassava are very limited. To extend our understanding of Hsfs in cassava, genome‐wide identification of MeHsfs was systematically analysed in this study, especially the involvement of MeHsf3 and salicylic acid (SA) signalling in the defence response.

Results

Comprehensive identification and bioinformatic analysis of MeHsfs

After confirmation using the conserved domain database (CDD) and Pfam database, detailed information on the predicted MeHsfs was obtained from the cassava (Manihot esculenta) annotation database at Phytozome v10.3 (http://www.phytozome.net/cassava.php) and is listed in Table S1 (Supporting Information), including the locus name, chromosome location, gene length, molecular weight (MW) and pI of the coding proteins.

To investigate the evolutionary link amongst 32 MeHsfs, 22 AtHsfs and 25 OsHsfs, the unrooted phylogenetic tree of these Hsfs was constructed (Fig. 1). The phylogenetic analysis indicated a closer evolution between MeHsfs and AtHsfs than OsHsfs. Moreover, the upstream exon–downstream intron characteristics and conserved motifs of MeHsfs, as well as their relation to phylogenetic links, were also analysed (Figs S1 and S2, see Supporting Information). These results showed that the exon lengths and numbers of 32 MeHsfs, as well as the conserved motifs, were directly related to their phylogenetic relationships (Figs S1 and S2).

Figure 1.

Figure 1

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The phylogenetic tree of 32 MeHsfs (Manihot esculenta heat stress transcription factors), 21 AtHsfs (Arabidopsis thaliana heat stress transcription factors) and 25 OsHsfs (Oryza sativa heat stress transcription factors). The unrooted phylogenetic tree was constructed using Clustalx 1.83 and mega5.05 software by the neighbour‐joining method. Red circles represent the Hsfs from cassava, blue triangles represent the Hsfs from rice and green squares represent the Hsfs from Arabidopsis.

Expression analysis of MeHsfs in response to flg22 and Xam

To investigate the possible involvement of MeHsfs in the plant defence response, we analysed the transcripts of these MeHsfs in response to flg22 and Xam treatments. Through quantitative real‐time polymerase chain reaction (PCR), we found that the transcript levels of 32 MeHsfs were commonly regulated after flg22 and Xam treatment for 1 and 6 h (Fig. S3, see Supporting Information), indicating the possible role of MeHsfs in the plant defence response. In response to Xam treatment, 13 MeHsfs (MeHsf3, 5, 6, 8, 10, 11, 12, 15, 16, 20, 21, 27, 30) were up‐regulated at 1 and 6 h. Amongst these genes, MeHsf3 showed the highest transcript level at 6 h. Although there is high homology between MeHsf3 and MeHsf4 (Fig. 1), the transcript level of MeHsf3 was higher at most time points in response to flg22 and Xam treatments in comparison with MeHsf4. Hence, MeHsf3 was chosen for further studies.

Subcellular localization and transcriptionally activated activity of MeHsf3

Herein, MeHsf3 was selected as the candidate for further investigation (Fig. 2A). Through transient expression in Nicotiana benthamiana leaves, we found that the fluorescence of 35S::green fluorescent protein (GFP) was located in both the cell nucleus and cytoplasm, whereas that of 35S::GFP‐MeHsf3 was specifically located in the cell nucleus, as evidenced by the co‐localization of 4′,6‐diamidino‐2‐phenylindole (DAPI) staining (Fig. 2B).

Figure 2.

Figure 2

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The expression profile and subcellular localization of MeHsf3 (Manihot esculenta heat stress transcription factor 3). (A) The transcripts of MeHsf3 in response to Xanthomonas axonopodis pv. manihotis (Xam) treatment. For the gene expression assay, 30‐day‐old cassava leaves were syringe infiltrated with 10 mm MgCl2 (mock for Xam) or 108 colony‐forming units (cfu)/mL of Xam for 0, 1 and 6 h. Asterisks (*) indicate significant differences in comparison with 0 h at P < 0.05. (B) The subcellular localization of MeHsf3. The Agrobacterium tumefaciens strain harbouring GV3101 empty vector or the recombinant plasmids, as well as P19, was syringe infiltrated into Nicotiana benthamiana leaves for 2 days. Then, the green fluorescent protein (GFP) signals and 4′,6‐diamidino‐2‐phenylindole (DAPI)‐stained cell nuclei in the infiltrated area were detected using a confocal laser‐scanning microscope. Bar, 25 μm. DIC, differential interference contrast microscope. (C) The transcriptionally activated activity of MeHsf3. For the assay, the effector plasmid (pEGAD or MeHsf3‐pEGAD) and reporter plasmid (4 × GAAACTTC (HSE)‐pGreenII0800‐LUC or 4 × GACACACT (mHSE)‐pGreenII0800‐LUC) were co‐transformed into leaf protoplasts. Renilla luciferase (REN) was used as an internal reference for the transcriptionally activated analyses. Asterisks (*) indicate significant differences in comparison with vector treatment at P < 0.05.

Plant Hsfs widely recognize and bind HSEs (5′‐GAAnnTTC‐3′). Thus, we performed dual luciferase (LUC) assay to investigate the effect of MeHsf3 overexpression on the activities of HSEs and mutated HSEs (mHSEs, 5′‐GACnnACT‐3′). We found that MeHsf3 overexpression significantly increased the LUC activity of 4 × GAAACTTC (HSE)‐pGreenII0800‐LUC, but had no significant effect on that of 4 × GACACACT (mHSE)‐pGreenII0800‐LUC), in cassava leaf protoplasts (Fig. 2C).

MeHsf3 is essential for the plant immune response to cassava bacterial blight

Virus‐induced gene silencing (VIGS) has been used extensively for the investigation of the in vivo roles of cassava genes in cassava (Wei et al., 2017a). MeHsf3‐silenced cassava plants (through VIGS) led to lower transcript levels of MeHsf3 (Fig. 3A). MeHsf3‐silenced cassava plants showed significantly higher bacterial numbers than those of empty vector plants at 4 days post‐inoculation (dpi). The lesion area was markedly larger after Xam syringe infiltration into the leaves of MeHsf3‐silenced cassava plants in comparison with that in empty vector plants at 6 dpi (Fig. 3B). These results suggest that MeHsf3 regulates plant susceptibility to cassava bacterial blight.

Figure 3.

Figure 3

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MeHsf3 (Manihot esculenta heat stress transcription factor 3) is essential for the plant immune response to cassava bacterial blight. (A) The transcript of MeHsf3 in MeHsf3‐silenced cassava leaves. (B) The bacterial number in MeHsf3‐silenced cassava leaves. Thirty‐day‐old cassava leaves were first syringe infiltrated with the GV3101 strain containing the virus‐induced gene silencing (VIGS) vector for 14 days; thereafter, the other cassava leaves were syringe infiltrated with 108 colony‐forming units (cfu)/mL of Xanthomonas axonopodis pv. manihotis (Xam). Bar, 1 cm. dpi, days post‐inoculation. Asterisks (*) indicate significant differences in comparison with vector treatment at P < 0.05.

MeHsf3 is a transcriptional activator of MeEDS1 and MePR4

In MeHsf3‐silenced cassava plants, not only the transcript of MeHsf3, but also the transcripts of MeEDS1, MePR2 and MePR4, showed significantly lower levels in comparison with those in control plants, whereas those of MePR1 and MePR3 displayed no significant difference (Figs 3B and 4A). When MeHsf3‐pEGAD was transformed into cassava leaf protoplasts, the transcripts of MeHsf3, as well as MeEDS1 and MePR4, were strongly up‐regulated (Fig. 4B). Because HSEs were commonly distributed in the promoters of Enhanced Disease Susceptibility 1 (MeEDS1) and pathogen‐related gene 4 (MePR4), but not in those of other MePRs (MePR1, MePR2 and MePR3), we analysed the direct link between MeHsf3 and the promoter regions. As revealed by the dual LUC assay, MeHsf3 overexpression significantly activated the promoter activities of MeEDS1 and MePR4 in cassava leaf protoplasts (Fig. 4C). Furthermore, chromatin immunoprecipitation (ChIP)‐PCR showed that MeEDS1 and MePR4 promoter fragments, including HSEs, were significantly enriched in the 35S::GFP‐MeHsf3 immunoprecipitated chromatin pellet using anti‐GFP in cassava leaf protoplasts (Fig. 4D). Moreover, the binding of MeHsf3 to the corresponding probes, including HSEs, was confirmed by the gel shift bands in the electrophoretic mobility shift assay (EMSA) (Fig. 4E). Taken together, we conclude that MeHsf3 is a transcriptional activator of MeEDS1 and MePR4 in cassava.

Figure 4.

Figure 4

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MeHsf3 (Manihot esculenta heat stress transcription factor 3) is a transcriptional activator of MeEDS1 (Manihot esculenta Enhanced Disease Susceptibility 1) and MePR4 (Manihot esculenta pathogen‐related gene 4). (A) The transcript levels of MeEDS1 and MePRs in MeHsf3‐silenced plant leaves. (B) The transcript levels of MeEDS1 and MePRs in cassava leaf protoplasts transformed by pEGAD or MeHsf3‐pEGAD. (C) The effect of MeHsf3 overexpression on the promoter activities of MeEDS1 and MePR4 in cassava leaf protoplasts by a dual luciferase (LUC) reporter system. (D) The enrichment of MeHsf3 in the promoters of MeEDS1 and MePR4 by chromatin immunoprecipitation‐polymerase chain reaction (ChIP‐PCR). (E) The binding of MeHsfs to the heat stress elements (HSEs) of MeEDS1 and MePR4 promoter regions by electrophoretic mobility shift assay (EMSA). The wild‐type probes and mutated probes are shown. Asterisks (*) indicate significant differences in comparison with vector treatment at P < 0.05.

MeEDS1 and MePR4 positively regulate the plant immune response to cassava bacterial blight

Through quantitative real‐time PCR, we found that the transcript levels of MeEDS1 and MePR4 were commonly regulated after Xam treatment for 1 and 6 h (Fig. 5A), indicating the possible role of MeEDS1 and MePR4 in the plant defence response. Through transient expression in N. benthamiana leaves, we found that the fluorescence of 35S::GFP‐MeEDS1 and 35S::GFP‐MePR4 was located in both the cell nucleus and cytoplasm (Fig. 5B).

Figure 5.

Figure 5

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The expression profiles of MeEDS1 (Manihot esculenta Enhanced Disease Susceptibility 1) and MePR4 (Manihot esculenta pathogen‐related gene 4). (A) The transcripts of MeEDS1 and MePR4 in response to Xanthomonas axonopodis pv. manihotis (Xam) treatment. For the gene expression assay, 30‐day‐old cassava leaves were syringe infiltrated with 10 mm MgCl2 (mock for Xam) or 108 colony‐forming units (cfu)/mL of Xam for 0, 1 and 6 h. Asterisks (*) indicate significant differences in comparison with 0 h at P < 0.05. (B) Subcellular localization of MeEDS1 and MePR4. The Agrobacterium tumefaciens strain harbouring GV3101 empty vector or the recombinant plasmid, as well as P19, was syringe infiltrated into Nicotiana benthamiana leaves for 2 days. After staining with 1 µg/mL 4′,6‐diamidino‐2‐phenylindole (DAPI) for 1 h, the GFP signals, DAPI‐stained cell nuclei and plasma membrane (PM)‐mCherry in the infiltrated area were detected using a confocal laser‐scanning microscope. DIC, differential interference contrast microscope. Bar, 25 μm.

Through transient expression in N. benthamiana leaves, we found that overexpression of MeEDS1 and MePR4 resulted in lower bacterial propagation of Xam in the leaves (Fig. 6A), conferring an improved immune response. Quantitative real‐time PCR analysis showed that the levels of transcripts of MeEDS1 and MePR4 in the corresponding silenced plants were only 40%–50% of those in empty vector plants (Fig. 6B). Compared with empty vector plants, there were 16.5% and 8.5% increases in the number of bacteria in MeEDS1‐ and MePR4‐silenced plants, respectively, at 6 dpi. Consistently, the lesion areas of MeEDS1‐ and MePR4‐silenced plants were markedly larger than those of empty vector plants (Fig. 6C). These results indicate that MeEDS1 and MePR4 regulate plant susceptibility to cassava bacterial blight, similarly to MeHsf3.

Figure 6.

Figure 6

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MeEDS1 (Manihot esculenta Enhanced Disease Susceptibility 1) and MePR4 (Manihot esculenta pathogen‐related gene 4) positively regulate plant immune response to cassava bacterial blight. (A) Through transient expression in Nicotiana benthamiana leaves, overexpression of MeEDS1 and MePR4 resulted in lower bacterial propagation of Xanthomonas axonopodis pv. manihotis (Xam) in the leaves. Nicotiana benthamiana leaves were first syringe infiltrated with different transients for 2 days; thereafter, the transient expressing leaves were syringe infiltrated with 108 colony‐forming units (cfu)/mL of Xam at the indicated time points (dpi, days post‐inoculation). (B) The transcripts of MeEDS1 and MePR4 in gene‐silenced cassava leaves. (C) The bacterial number in MeEDS1‐ and MePR4‐silenced cassava leaves. The 30‐day‐old cassava leaves were first syringe infiltrated with GV3101 strain containing the virus‐induced gene silencing (VIGS) vector for 14 days; thereafter, the other cassava leaves were syringe infiltrated with 108 cfu/mL of Xam. Bar, 1 cm. Asterisks (*) indicate significant differences in comparison with vector treatment at P < 0.05.

Exogenous SA restores disease sensitivity of MeHsf3‐, MeEDS1‐ and MePR4‐silenced cassava plants

EDS1 is an important gene for SA biosynthesis (Shi et al., 2014). Therefore, we examined the endogenous SA level in MeHsf3‐, MeEDS1‐ and MePR4‐silenced cassava leaves. Consistent with the transcript of MeEDS1, MeHsf3‐ and MeEDS1‐silenced plants showed significantly lower SA levels, whereas MePR4‐silenced plants displayed no significant difference in comparison with the control (Fig. 7A). In addition, MeHsf3‐ and MeEDS1‐silenced plants displayed significantly lower transcript levels of MePR2 and MePR4, whereas MePR4‐silenced plants exhibited a lower transcript level of MePR4 (Fig. S4, see Supporting Information). After Xam infection, the bacterial numbers in MeHsf3‐, MeEDS1‐ and MePR4‐silenced cassava leaves were significantly greater than in the control (Fig. 7B,C). Thus, either a decreased SA level or a lower transcript level of MePR4 downstream of SA signalling results in disease sensitivity in cassava.

Figure 7.

Figure 7

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Exogenous salicylic acid (SA) restores the disease sensitivity of MeHsf3 (Manihot esculenta heat stress transcription factor 3)‐, MeEDS1 (Manihot esculenta Enhanced Disease Susceptibility 1)‐ and MePR4 (Manihot esculenta pathogen‐related gene 4)‐silenced cassava plants. (A) The endogenous SA level in gene‐silenced cassava leaves. FW, fresh weight. (B) The bacterial number in gene‐silenced cassava leaves. cfu, colony‐forming unit; dpi, days post‐inoculation. (C) The phenotype in gene‐silenced cassava leaves. Thirty‐day‐old cassava leaves were first syringe infiltrated with GV3101 strain containing the virus‐induced gene silencing (VIGS) vector for 14 days; thereafter, the cassava leaves were sprayed with 10 µm SA and syringe infiltrated with 108 cfu/mL of Xanthomonas axonopodis pv. manihotis (Xam). Bar, 1 cm. Different letters indicate significant differences in comparison with vector treatment at P < 0.05.

After SA pretreatment, the endogenous SA level was strongly increased with no significant difference amongst these plants (Fig. 7A). Similarly, the bacterial number in MeHsf3‐, MeEDS1‐ and MePR4‐silenced cassava leaves exhibited no significant difference compared with mock plants (Fig. 7B,C). Correspondingly, the transcript levels of MePR1–3 were strongly activated in all plants, with no significant differences in mock, MeHsf3‐silenced or MeEDS1‐silenced cassava plants (Fig. S4). Notably, the levels of the transcripts of MePR1 and MePR2 in MePR4‐silenced plants were much higher than those of the other plants (Fig. S4), and the results may contribute to the defence response in MePR4‐silenced lines on SA pretreatment. These results indicate that MeHsf3 may regulate plant susceptibility to cassava bacterial blight through the modulation of SA biosynthesis and downstream genes in SA signalling.

Discussion

As sessile organisms, plants cannot change their location and live in a fixed environment. When unfavourable and harsh stresses (abiotic and biotic stresses) are applied, plants must respond and cope with these stresses through the evolution of complex signalling pathways. In the plant–pathogen interaction, pathogen‐associated molecular pattern (PAMP)‐triggered immunity (PTI) and effector‐triggered immunity (ETI) play important roles in the plant defence response, resulting in reactive oxygen species (ROS) and reactive nitrogen species (RNS) accumulation, the hypersensitive response (HR) and the activation of expression of multiple defence genes (Bent and Mackey, 2007; Yun et al., 2011). SA signalling is a key component of both PTI and ETI. In response to pathogen infection, plant endogenous SA, ROS and RNS are quickly and strongly induced; thereafter, the monomerization of NPR1 is triggered with further translocation to the nucleus. Then, nuclear NPR1 physically interacts with the TGACG sequence‐specific binding protein (TGA), which binds to the promoters of downstream genes of SA signalling, such as PR1 (Yun et al., 2011).

In plant stress responses, transcription factors play essential roles by linking the upstream protein kinase and downstream gene expression. As the terminal components of signal transduction, plant Hsfs mediate the activation of multiple genes responsive to various stresses (Huang et al., 2015; Singh et al., 2012; Yabuta, 2016). However, information and functional analysis are very limited in non‐model plants, especially in cassava (Manihot esculenta), one of the most important staple crops in tropical areas. In this study, 32 MeHsfs were identified in a genome‐wide analysis in cassava. The transcript levels of several MeHsfs were commonly regulated after flg22 and Xam treatment. In response to Xam treatment, 13 MeHsfs (MeHsf3, 5, 6, 8, 10, 11, 12, 15, 16, 20, 21, 27 and 30) were up‐regulated at 1 and 6 h post‐inoculation (hpi). Amongst these genes, MeHsf3 showed the highest transcript level at 6 hpi. SlHsfA1a, markedly induced by root‐knot nematode (RKN) treatment, positively regulates RKN resistance in tomato (Zhou et al., 2018). Transcription activator‐like effectors were also induced by Xam 668 in cassava (Cohn et al., 2014). Hence, MeHsf3 was chosen as a candidate for further investigation. The specific subcellular location in the nucleus and the transcriptional activation of HSE confirmed that MeHsf3 is a transcription factor that can recognize and bind HSEs.

Further investigation showed that MeHsf3, MeEDS1 and MePR4 regulate plant susceptibility to cassava bacterial blight, as evidenced by bacterial propagation after Xam syringe infiltration in cassava leaves with modulated gene expression (through VIGS). Zhou et al. (2018) have demonstrated that SlHsfA1a plays a significant role in RKN resistance by regulation of H2O2 production via Tobacco rattle virus (TRV) silencing and overexpression. Multiple studies have certified that PRs play crucial roles in the resistance of plants to various biotic and abiotic stresses (Schultheiss et al., 2003). NtPR‐Q, a member of the PR‐3 family, positively regulates the tolerance to Ralstonia solanacearum attack in tobacco (Tang et al., 2017). Similarly, PR10 is involved in the plant response to microbial pathogens, such as Colletotrichum acutatum, Xanthomonas campestris pv. vesicatoria, Tobacco mosaic virus, Pseudomonas syringae pv. tomato and Hyaloperonospora arabidopsidis in pepper (Choi et al., 2012; Park et al., 2004; Soh et al., 2012). Notably, as positive regulators of defence resistance, dual LUC assay, ChIP‐PCR and EMSA confirmed that MeEDS1 and MePR4 were direct targets of MeHsf3. In addition to the TGA of the basic domain‐leucine zipper (bZIP) transcription factor, which can bind to the promoters of PR1, whitefly induced 1 and autophagy‐related genes (Atg10 and Atg18f) (Alves et al., 2013; Wang et al., 2015; Zhou et al., 2018), plant Hsfs may also function as transcriptional activators of PRs. This study extends our understanding of MeHsf3, especially its involvement in defence resistance as well as transcriptional activation of MePR4.

The eds1 mutant was first identified via the suppression of R‐gene‐mediated resistance to an oomycete pathogen in Arabidopsis (Parker et al., 1996). Further studies have shown that AtEDS1 is an essential component of multiple R‐gene‐mediated disease resistances in Arabidopsis, and AtEDS1 operates upstream of SA accumulation and the SA‐dependent defence response (Falk et al., 1999; Feys et al., 2001; Kim et al., 2012; Wiermer et al., 2005). In addition to MePR4, MeEDS1 has also been identified as a direct target of MeHsf3. Moreover, MeHsf3‐ and MeEDS1‐silenced plants show significantly lower SA levels, as well as transcript levels of MePRs, and exogenous SA restores the disease sensitivity of MeHsf3‐, MeEDS1‐ and MePR4‐silenced cassava plants. PRs function downstream of SA signalling. In this study, a decreased SA level or lower transcript level of MePR4, without affecting the endogenous SA level, resulted in disease sensitivity, highlighting the essential role of MePR4 in the immune response of cassava. After pretreatment with SA, the transcript levels of MePRs were strongly activated in MeHsf3‐, MeEDS1‐ and MePR4‐silenced cassava plants, as well as vector transformed leaves, especially the transcripts of MePR1 and MePR2 in MePR4‐silenced lines. We noticed that the transcripts of PR1–3 showed no significant differences in mock plants and MeHsf3‐ and MeEDS1‐silenced cassava plants. These results indicate that higher levels of transcripts of MePR1 and MePR2 may contribute to the defence response in MePR4‐silenced lines on SA pretreatment. Hence, we conclude that MeHsf3 may regulate the plant immune response through a two‐pronged pathway: (i) MeHsf3 activates MeEDS1 leading to SA accumulation; (ii) MeHsf3 activates the transcript of MePR4, resulting in a direct immune response (Fig. 8).

Figure 8.

Figure 8

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The proposed model of the MeHsf3 (Manihot esculenta heat stress transcription factor 3)‐mediated plant immune response in cassava. SA, salicylic acid.

Taken together, this is the first study to show the essential role of MeHsf3 in the plant immune response to cassava bacterial blight through the regulation of the transcript levels of MeEDS1 and MePR4.

Experimental Procedures

Plant materials and growth conditions

The South China 124 (SC124) variety of cassava was used in this study. The plants were cultivated in soil in a glasshouse, which was controlled at 12 h light/12 h dark and 28 °C light/26 °C dark, with irradiance of 120–150 µmol quanta/m2/s. The soil was watered with Hoagland's solution twice every week to maintain the growth of cassava plants.

Comprehensive characterization and bioinformatic analysis of MeHsfs

The sequences of MeHsfs were searched for and obtained from Phytozome v10.3 (http://www.phytozome.net/cassava.php), and confirmed using CDD (http://www.ncbi.nlm.nih.gov/cdd) (Marchler‐Bauer et al., 2015) and the Pfam database (http://pfam.xfam.org) (Finn et al., 2016). The predicted MW and theoretical pI of the coding proteins were analysed by ProtParam software (http://web.expasy.org/protparam). After obtaining the sequences of AtHsfs and OsHsfs from The Arabidopsis Information Resource (TAIR) version 10 (http://www.Arabidopsis.org) and Rice Genome Annotation Project (RGAP) version 7 (http://rice.plantbiology.msu.edu), respectively, we constructed the neighbour‐joining phylogenetic tree of MeHsfs using mega5.05 and Clustalx 1.83 software (Tamura et al., 2011).

Moreover, the gene structures of MeHsfs were analysed using the Gene Structure Display Server (GSDS) v2.0 (http://gsds.cbi.pku.edu.cn/index.php) (Hu B et al., 2015), and the conserved motifs of MeHsfs were identified using Multiple Em for Motif Elicitation (MEME) v4.11.0 (http://meme-suite.org/tools/meme).

RNA isolation and quantitative real‐time PCR

Total RNA was extracted using an RNAprep Pure Plant Kit (DP441, TIANGEN, Beijing, China), and the contaminating DNA was removed using RNase‐free DNase (M0303S, NEB, Ipswich, Massachusetts, USA). Thereafter, a RevertAid First Strand cDNA Synthesis Kit (K1622, Thermo Scientific, Waltham, MA, USA) was used for first‐strand cDNA synthesis from mRNA according to the manufacturer's instructions. The cDNA was quantified and diluted five times, and 2 µL of cDNA was used as template in every 20‐µL reaction mixture employing FastStart Essential DNA Green Master (06924204001, Roche, Basel, Switzerland) in a LightCycler® 96 Real‐Time PCR System (Roche). With the values of Ct and the reference gene of MeEF1a, the relative gene expression levels were analysed using the comparative ΔΔCt method. The primers used for quantitative real‐time PCR are listed in Table S2 (see Supporting Information).

Vector construction and transient expression in N. benthamiana leaves

The coding sequences of MeHsf3, MeEDS1 and MePR4 were amplified by PCR, verified by DNA sequencing and cloned into pEGAD (Cutler et al., 2000) to generate N‐terminal GFP fusion constructs. The corresponding primers are listed in Table S3 (see Supporting Information). The Agrobacterium tumefaciens strain harbouring the GV3101 empty vector or the recombinant plasmids, as well as P19, was syringe infiltrated into N. benthamiana leaves as described by Sparkes et al. (2006). At 2 dpi, after staining with 1 µg/mL DAPI for 1 h, the GFP signals, DAPI‐stained cell nuclei and plasma membrane (PM)‐mCherry in the infiltrated area were detected using a confocal laser‐scanning microscope (TCS SP8, Leica, Heidelberg, Germany) in the transformed N. benthamiana leaves.

VIGS in cassava

The partial coding sequences of MeHsf3, MeEDS1 and MePR4 were amplified by PCR, verified by DNA sequencing and cloned into pTRV2 (Liu et al., 2002). The corresponding primers are listed in Table S3. The Agrobacterium tumefaciens strain GV3101 harbouring pTRV2 or the recombinant plasmid, together with pTRV1, was syringe infiltrated into cassava leaves as described by Wei et al. (2017a). At 14 dpi, the corresponding gene expression and disease assay at different time points as indicated were analysed in cassava leaves.

Xam infiltration

After shaking in 10 mL of Luria‐Bertani (LB) liquid medium at 28 °C for 12 h, 1 mL of Xam culture was transferred to new LB liquid culture and shaken to an optical density at 600 nm (OD600) of 0.6. Then, the Xam culture was centrifuged and diluted to 108 colony‐forming units (cfu)/mL using 10 mm MgCl2 and 0.05% silwet L‐77, and syringe infiltrated into the abaxial side of plant leaves as described by Wei et al. (2017a). At the indicated time points after growth in a glasshouse, at least 15 leaves in every biological repeat were harvested for the quantification of bacterial populations.

Transient expression assay of LUC reporter gene

For the constructs, the promoter regions of MeEDS1 and MePR4 were amplified by PCR, verified by DNA sequencing and cloned into pGreenII0800‐LUC. The primers are listed in Table S3. Then, the purified plasmid together with empty vector (pEGAD) and MeHsf3‐pEGAD were transfected into protoplasts as described by Yoo et al. (2007). The firefly LUC and renilla LUC in the protoplasts were assayed using a Dual Luciferase Reporter Gene Assay Kit (RG027, Beyotime, Haimen City, Jiangsu Province, China) according to the manufacturer's instructions.

For the assay of the transcriptionally activated activity of MeHsf3, the effector plasmid (pEGAD or MeHsf3‐pEGAD) and reporter plasmid (4 × GAAACTTC (HSE)‐mini35S‐pGreenII0800‐LUC or 4 × GACACACT (mHSE)‐mini35S‐pGreenII0800‐LUC) were co‐transformed into leaf protoplasts as described by Yoo et al. (2007).

ChIP

The purified empty vector (pEGAD) and MeHsf3‐pEGAD were transfected into protoplasts as described by Yoo et al. (2007). After 12 h, the protoplasts were used for chromatin pellet extraction and nuclease digestion as described previously by O'Neill and Turner (2003). After immunoprecipitation by the anti‐GFP antibody (AG281, Beyotime), the MeHsf3–DNA complex was used for quantitative real‐time PCR to analyse the DNA fragment enrichment.

EMSA analysis

EMSA analysis was performed as described by Ream et al. (2016). For the construction of the recombinant protein, the coding sequence of MeHsf3 was amplified by PCR, verified by DNA sequencing and cloned into pET28a. The primers are listed in Table S3. The recombinant proteins were expressed and induced in Escherichla coli strain BL21 (DE3) as described by Wang et al. (2015). About 20 ng of double‐stranded probes and 10 µg of protein in a mixture of 50 µL were vortexed and incubated at 25 °C for 2 h. After incubation, 20 µL of mixture were loaded into each well in 2% (w/v) agarose gel using 0.5 × TB buffer (45 mm Tris, 45 mm boric acid). After staining with ethidium bromide (EB), the agarose gel was visualized using a GelDoc imager system (BIO‐RAD, Hercules, CA, USA).

Quantification of endogenous SA level

The endogenous SA level in plant leaves was extracted and examined as described previously by Shi et al. (2014) and Wei et al. (2017b).

Statistical analysis

All experiments were performed with at least three biological replicates, and the data are shown as average means with standard deviations (SDs). Significant differences between different constructs are shown as asterisks (*) at P < 0.05 using analysis of variance (ANOVA) and Student's t‐test.

Supporting information

Additional Supporting Information may be found in the online version of this article at the publisher's website:

Fig. S1 The gene structure of 32 MeHsfs (Manihot esculenta heat stress transcription factors). The analysis of the phylogenetic relationship and gene structure of 32 MeHSFs using mega5.05 software and GSDS v2.0.

Click here for additional data file. (448.9KB, tif)

Fig. S2 The conserved motif analysis of 32 MeHsfs (Manihot esculenta heat stress transcription factors). Link analysis of the phylogenetic relationship and conserved motifs of 32 MeHSFs using mega5.05 software and MEME v4.11.0.

Click here for additional data file. (2.1MB, tif)

Fig. S3 The transcripts of 32 MeHsfs (Manihot esculenta heat stress transcription factors) in response to different treatments. For the gene expression assay, 30‐day‐old cassava leaves were sprayed with water (mock for flg22) or 10 μm flg22, or syringe infiltrated with 10 mm MgCl2 [mock for Xanthomonas axonopodis pv. manihotis (Xam)] or 108 colony‐forming units (cfu/mL) of Xam for 0, 1 and 6 h. Asterisks (*) indicate significant differences in comparison with mock treatment at P < 0.05.

Click here for additional data file. (220.9KB, png)

Fig. S4 The transcripts of MePRs (Manihot esculenta pathogen‐related genes) in gene‐silenced cassava leaves under mock conditions and pretreatment with salicylic acid (SA). Thirty‐day‐old cassava leaves were first syringe infiltrated with GV3101 strain containing the virus‐induced gene silencing (VIGS) vector for 14 days; thereafter, cassava leaves were sprayed with 10 µm salicylic acid (SA) and syringe infiltrated with 108 colony‐forming units (cfu)/mL of Xanthomonas axonopodis pv. manihotis (Xam). Different letters indicate significant differences in comparison with vector treatment at P < 0.05.

Click here for additional data file. (349.7KB, png)

Table S1 Identification of heat stress transcription factors (Hsfs), Enhanced Disease Susceptibility 1 (EDS1) and pathogen‐related genes (PRs) from cassava (Manihot esculenta).

Click here for additional data file. (25.4KB, docx)

Table S2 The primers used in quantitative real‐time polymerase chain reaction.

Click here for additional data file. (27.4KB, docx)

Table S3 The primers used in vector construction.

Click here for additional data file. (22.6KB, docx)

Acknowledgements

We thank Dr Chris R. Somerville, Dr Yanru Hu, Dr Jie Zhou and Dr Jiang Tian for sharing their vector plasmids. This research was supported by the National Natural Science Foundation of China (No. 31760067) and the Startup Funding and Scientific Research Foundation of Hainan University (No. kyqd1531) to Haitao Shi.

References

  1. Almoguera, C. , Personat, J.M. , Prieto‐Dapena, P. and Jordano, J. (2015) Heat shock transcription factors involved in seed desiccation tolerance and longevity retard vegetative senescence in transgenic tobacco. Planta, 242, 461–475. [DOI] [PubMed] [Google Scholar]
  2. Alves, M.S. , Dadalto, S.P. , Gonçalves, A.B. , De Souza, G.B. , Barros, V.A. and Fietto, L.G. (2013) Plant bZIP transcription factors responsive to pathogens: a review. Int. J. Mol. Sci. 14, 7815–7828. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bechtold, U. , Albihla, lWS. , Lawson, T. , Fryer, M.J. , Sparrow, P.A. , Richard, F. , Persad, R. , Bowden, L. , Hickman, R. , Martin, C. , Beynon, J.L. , Buchanan‐Wollaston, V. , Baker, N.R. , Morison, J.I. , Schöffl, F. , Ott, S. and Mullineaux, P.M. (2013) Arabidopsis HEAT SHOCK TRANSCRIPTION FACTORA1b overexpression enhances water productivity, resistance to drought, and infection. J. Exp. Bot. 64, 3467–3481. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bent, A.F. and Mackey, D. (2007) Elicitors, effectors, and R genes: the new paradigm and a lifetime supply of questions. Annu. Rev. Phytopathol. 45, 399–436. [DOI] [PubMed] [Google Scholar]
  5. Boher, B. , Kpemoua, L. , Nicole, M. , Luisetii, J. and Geiger, J.P. (1995) Ultrastructure of interaction between cassava and Xanthomonas axonopodis pv manihotis: cytochemistry of cellulose and pectin degradation in a susceptible cultivar. Am. Phytopathol. Soc. 85, 777–788. [Google Scholar]
  6. Charng, Y.Y. , Liu, H.C. , Liu, N.Y. , Chi, W.T. , Wang, C.N. , Chang, S.H. and Wang, T.T. (2007) A heat‐inducible transcription factor, HsfA2, is required for extension of acquired thermotolerance in Arabidopsis . Plant Physiol. 143, 251–262. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chauhan, H. , Khurana, N. , Agarwal, P. and Khurana, P. (2011) Heat shock factors in rice (Oryza sativa L.): genome‐wide expression analysis during reproductive development and abiotic stress. Mol. Genet. Genomics, 286, 171–187. [DOI] [PubMed] [Google Scholar]
  8. Chauhan, H. , Khurana, N. , Agarwal, P. , Khurana, J.P. and Khurana, P. (2013) A seed preferential heat shock transcription factor from wheat provides abiotic stress tolerance and yield enhancement in transgenic Arabidopsis under heat stress environment. PLoS One, 8, e79577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cheng, Q. , Zhou, Y. , Liu, Z. , Zhang, L. , Song, G. , Guo, Z. , Wang, W. , Qu, X. , Zhu, Y. and Yang, D. (2015) An alternatively spliced heat shock transcription factor, OsHSFA2dI, functions in the heat stress‐induced unfolded protein response in rice. Plant Biol. 17, 419–429. [DOI] [PubMed] [Google Scholar]
  10. Choi, D.S. , Hwang, I.S. and Hwang, B.K. (2012) Requirement of the cytosolic interaction between PATHOGENESIS‐RELATED PROTEIN10 and LEUCINE‐RICH REPEAT PROTEIN1 for cell death and defense signaling in pepper. Plant Cell, 24, 1675–1690. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chung, E. , Kim, K.M. and Lee, J.H. (2013) Genome‐wide analysis and molecular characterization of heat shock transcription factor family in Glycine max . J. Genet. Genomics, 40, 127–135. [DOI] [PubMed] [Google Scholar]
  12. Cohn, M. , Bart, R.S. , Shybut, M. , Dahlbeck, D. , Gomez, M. , Morbitzer, R. , Hou, B.H. , Frommer, W.B. , Lahaye, T. and Staskawicz, B.J. (2014) Xanthomonas axonopodis virulence is promoted by a transcription activator‐like effector‐mediated induction of a SWEET sugar transporter in cassava. Mol. Plant–Microbe Interact. 27, 1186–1198. [DOI] [PubMed] [Google Scholar]
  13. Cutler, S.R. , Ehrhardt, D.W. , Griffitts, J.S. and Somerville, C.R. (2000) Random GFP::cDNA fusions enable visualization of subcellular structures in cells of Arabidopsis at a high frequency. Proc. Natl. Acad. Sci. USA, 97, 3718–3723. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Falk, A. , Feys, B.J. , Frost, L.N. , Jones, J.D. , Daniels, M.J. and Parker, J.E. (1999) EDS1, an essential component of R gene‐mediated disease resistance in Arabidopsis has homology to eukaryotic lipases. Proc. Natl. Acad. Sci. USA, 96, 3292–3297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Feys, B.J. , Moisan, L.J. , Newman, M.A. and Parker, J.E. (2001) Direct interaction between the Arabidopsis disease resistance signaling proteins, EDS1 and PAD4. EMBO J. 20, 5400–5411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Finn, R.D. , Coggill, P. , Eberhardt, R.Y. , Eddy, S.R. , Mistry, J. , Mitchell, A.L. , Potter, S.C. , Punta, M. , Qureshi, M. , Sangrador‐Vegas, A. , Salazar, G.A. , Tate, J. and Bateman, A. (2016) The Pfam protein families database: towards a more sustainable future. Nucleic Acids Res. 44, D279–D285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Friedberg, J.N. , Bowley, S.R. , McKersie, B.D. , Gurley, W.B. and Czarnecka‐Verner, E. (2006) Isolation and characterization of class A4 heat shock transcription factor from alfalfa. Plant Sci. 171, 332–344. [DOI] [PubMed] [Google Scholar]
  18. Giesguth, M. , Sahm, A. , Simon, S. and Dietz, K.J. (2015) Redox‐dependent translocation of the heat shock transcription factor AtHSFA8 from the cytosol to the nucleus in Arabidopsis thaliana . FEBS Lett. 589, 718–725. [DOI] [PubMed] [Google Scholar]
  19. Guo, J. , Wu, J. , Ji, Q. , Wang, C. , Luo, L. , Yuan, Y. , Wang, Y. and Wang, J. (2008) Genome‐wide analysis of heat shock transcription factor families in rice and Arabidopsis . J. Genet. Genomics, 35, 105–118. [DOI] [PubMed] [Google Scholar]
  20. Guo, M. , Yin, Y.X. , Ji, J.J. , Ma, B.P. , Lu, M.H. and Gong, Z.H. (2014) Cloning and expression analysis of heat‐shock transcription factor gene CaHsfA2 from pepper (Capsicum annuum L.). Genet. Mol. Res. 13, 1865–1875. [DOI] [PubMed] [Google Scholar]
  21. Guo, M. , Lu, J.P. , Zhai, Y.F. , Chai, W.G. , Gong, Z.H. and Lu, M.H. (2015) Genome‐wide analysis, expression profile of heat shock factor gene family (CaHsfs) and characterisation of CaHsfA2 in pepper (Capsicum annuum L.). BMC Plant Biol. 15, 151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hu, B. , Jin, J. , Guo, A.Y. , Zhang, H. , Luo, J. and Gao, G. (2015) GSDS 2.0: an upgraded gene feature visualization server. Bioinformatics, 31, 1296–1297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hu, Y. , Han, Y.T. , Wei, W. , Li, Y.J. , Zhang, K. , Gao, Y.R. , Zhao, F.L. and Feng, J.Y. (2015) Identification, isolation, and expression analysis of heat shock transcription factors in the diploid woodland strawberry Fragaria vesca . Front. Plant Sci. 6, 736. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hu, Y. , Han, Y.T. , Zhang, K. , Zhao, F.L. , Li, Y.J. , Zheng, Y. , Wang, Y.J. and Wen, Y.Q. (2016) Identification and expression analysis of heat shock transcription factors in the wild Chinese grapevine (Vitis pseudoreticulata). Plant Physiol. Biochem. 99, 1–10. [DOI] [PubMed] [Google Scholar]
  25. Huang, X.Y. , Tao, P. , Li, B.Y. , Wang, W.H. , Yue, Z.C. , Lei, J.L. and Zhong, X.M. (2015) Genome‐wide identification, classification, and analysis of heat shock transcription factor family in Chinese cabbage (Brassica rapa pekinensis). Genet. Mol. Res. 14, 2189–2204. [DOI] [PubMed] [Google Scholar]
  26. Hwang, S.M. , Kim, D.W. , Woo, M.S. , Jeong, H.S. , Son, Y.S. , Akhter, S. , Choi, G.J. and Bahk, J.D. (2014) Functional characterization of Arabidopsis HsfA6a as a heat‐shock transcription factor under high salinity and dehydration conditions. Plant Cell Environ. 37, 1202–1222. [DOI] [PubMed] [Google Scholar]
  27. Jin, G.H. , Gho, H.J. and Jung, K.H. (2013) A systematic view of rice heat shock transcription factor family using phylogenomic analysis. J. Plant Physiol. 170, 321–329. [DOI] [PubMed] [Google Scholar]
  28. Kim, T.H. , Kunz, H.H. , Bhattacharjee, S. , Hauser, F. , Park, J. , Engineer, C. , Liu, A. , Ha, T. , Parker, J.E. , Gassmann, W. and Schroeder, J.I. (2012) Natural variation in small molecule‐induced TIR‐NB‐LRR signaling induces root growth arrest via EDS1‐ and PAD4‐complexed R protein VICTR in Arabidopsis . Plant Cell, 24, 5177–5192. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kotak, S. , Port, M. , Ganguli, A. , Bicker, F. and von Koskull‐Döring, P. (2004) Characterization of C‐terminal domains of Arabidopsis heat stress transcription factors (Hsfs) and identification of a new signature combination of plant class A Hsfs with AHA and NES motifs essential for activator function and intracellular localization. Plant J. 39, 98–112. [DOI] [PubMed] [Google Scholar]
  30. Li, P.S. , Yu, T.F. , He, G.H. , Chen, M. , Zhou, Y.B. , Chai, S.C. , Xu, Z.S. and Ma, Y.Z. (2014) Genome‐wide analysis of the Hsf family in soybean and functional identification of GmHsf‐34 involvement in drought and heat stresses. BMC Genomics, 15, 1009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lin, Y. , Cheng, Y. , Jin, J. , Jin, X. , Jiang, H. , Yan, H. and Cheng, B. (2014) Genome duplication and gene loss affect the evolution of heat shock transcription factor genes in legumes. PLoS One, 9, e102825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lin, Y.X. , Jiang, H.Y. , Chu, Z.X. , Tang, X.L. , Zhu, S.W. and Cheng, B.J. (2011) Genome‐wide identification, classification and analysis of heat shock transcription factor family in maize. BMC Genomics, 12, 76. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Liu, Y. , Schiff, M. and Dinesh‐Kumar, S.P. (2002) Virus‐induced gene silencing in tomato. Plant J. 31, 777–786. [DOI] [PubMed] [Google Scholar]
  34. Liu, Z.W. , Wu, Z.J. , Li, X.H. , Huang, Y. , Li, H. , Wang, Y.X. and Zhuang, J. (2016) Identification, classification, and expression profiles of heat shock transcription factors in tea plant (Camellia sinensis) under temperature stress. Gene, 576, 52–59. [DOI] [PubMed] [Google Scholar]
  35. Marchler‐Bauer, A. , Derbyshire, M.K. , Gonzales, N.R. , Lu, S. , Chitsaz, F. , Geer, L.Y. , Geer, R.C. , He, J. , Gwadz, M. , Hurwitz, D.I. , Lanczycki, C.J. , Lu, F. , Marchler, G.H. , Song, J.S. , Thanki, N. , Wang, Z. , Yamashita, R.A. , Zhang, D. , Zheng, C. and Bryant, S.H. (2015) CDD: NCBI's conserved domain database. Nucleic Acids Res. 43, D222–D226. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Nishizawa, A. , Yabuta, Y. , Yoshida, E. , Maruta, T. , Yoshimura, K. and Shigeoka, S. (2006) Arabidopsis heat shock transcription factor A2 as a key regulator in response to several types of environmental stress. Plant J. 48, 535–547. [DOI] [PubMed] [Google Scholar]
  37. Nishizawa‐Yokoi, A. , Nosaka, R. , Hayashi, H. , Tainaka, H. , Maruta, T. , Tamoi, M. , Ikeda, M. , Ohme‐Takagi, M. , Yoshimura, K. , Yabuta, Y. and Shigeoka, S. (2011) HsfA1d and HsfA1e involved in the transcriptional regulation of HsfA2 function as key regulators for the Hsf signaling network in response to environmental stress. Plant Cell Physiol. 52, 933–945. [DOI] [PubMed] [Google Scholar]
  38. Okogbenin, E. , Setter, T. , Ferguson, M. , Mutegi, R. , Ceballos, H. , Olasanmi, B. and Fregene, M. (2013) Phenotypic approaches to drought in cassava: review. Front. Physiol. 4, 1–15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. O'Neill, L.P. and Turner, B.M. (2003) Immunoprecipitation of native chromatin: NChIP. Methods, 31, 76–82. [DOI] [PubMed] [Google Scholar]
  40. Park, C.J. , Kim, K.J. , Shin, R. , Park, J.M. , Shin, Y.C. and Paek, K.H. (2004) Pathogenesis‐related protein 10 isolated from hot pepper functions as a ribonuclease in an antiviral pathway. Plant J. 37, 186–198. [DOI] [PubMed] [Google Scholar]
  41. Parker, J.E. , Holub, E.B. , Frost, L.N. , Falk, A. , Gunn, N.D. and Daniels, M.J. (1996) Characterization of eds1, a mutation in Arabidopsis suppressing resistance to Peronospora parasitica specified by several different RPP genes. Plant Cell, 8, 2033–2046. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Qiao, X. , Li, M. , Li, L. , Yin, H. , Wu, J. and Zhang, S. (2015) Genome‐wide identification and comparative analysis of the heat shock transcription factor family in Chinese white pear (Pyrus bretschneideri) and five other Rosaceae species. BMC Plant Biol. 15, 12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Ream, J.A. , Lewis, L.K. and Lewis, K.A. (2016) Rapid agarose gel electrophoretic mobility shift assay for quantitating protein:RNA interactions. Anal. Biochem. 511, 36–41. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Schultheiss, H. , Dechert, C. , Király, L. , Fodor, J. , Michel, K. and Kogel, K.‐H. (2003) Functional assessment of the pathogenesis‐related protein PR‐1b in barley. Plant Sci. 165, 1275–1280. [Google Scholar]
  45. Shen, Z. , Yao, J. , Sun, J. , Chang, L. , Wang, S. , Ding, M. , Qian, Z. , Zhang, H. , Zhao, N. , Sa, G. Hou, P. , Lang, T. , Wang, F. , Zhao, R. , Shen, X. , and Chen, S. (2015) Populus euphratica HSF binds the promoter of WRKY1 to enhance salt tolerance. Plant Sci 235, 89–100. [DOI] [PubMed] [Google Scholar]
  46. Shi, H. , Wang, X. , Ye, T. , Chen, F. , Deng, J. , Yang, P. , Zhang, Y. and Chan, Z. (2014) The Cysteine2/Histidine2‐type transcription factor ZINC FINGER OF ARABIDOPSIS THALIANA 6 modulates biotic and abiotic stress responses by activating salicylic acid‐related genes and C‐REPEAT‐BINDING FACTOR genes in Arabidopsis . Plant Physiol. 165, 1367–1379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Shim, D. , Hwang, J.U. , Lee, J. , Lee, S. , Choi, Y. , An, G. , Martinoia, E. and Lee, Y. (2009) Orthologs of the class A4 heat shock transcription factor HsfA4a confer cadmium tolerance in wheat and rice. Plant Cell, 21, 4031–4043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Singh, A. , Mittal, D. , Lavania, D. , Agarwal, M. , Mishra, R.C. and Grover, A. (2012) OsHsfA2c and OsHsfB4b are involved in the transcriptional regulation of cytoplasmic OsClpB (Hsp100) gene in rice (Oryza sativa L.). Cell Stress Chaperones, 17, 243–254. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Soh, H.C. , Park, A.R. , Park, S. , Back, K. , Yoon, J.B. , Park, H.G. and Kim, Y.S. (2012) Comparative analysis of pathogenesis‐related protein 10 (PR10) genes between fungal resistant and susceptible peppers. Eur. J. Plant Pathol. 132, 37–48. [Google Scholar]
  50. Song, X. , Liu, G. , Duan, W. , Liu, T. , Huang, Z. , Ren, J. , Li, Y. and Hou, X. (2014) Genome‐wide identification, classification and expression analysis of the heat shock transcription factor family in Chinese cabbage. Mol. Genet. Genomics, 289, 541–551. [DOI] [PubMed] [Google Scholar]
  51. Sorger, P.K. and Pelham, H.R.B. (1988) Yeast heat‐shock factor is an essential DNA‐binding protein that exhibits temperature‐dependent phosphorylation. Cell, 54, 855–864. [DOI] [PubMed] [Google Scholar]
  52. Sparkes, I.A. , Runions, J. , Kearns, A. and Hawes, C. (2006) Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants. Nat. Protoc. 1, 2019–2025. [DOI] [PubMed] [Google Scholar]
  53. Stief, A. , Brzezinka, K. , Lämke, J. and Bäurle, I. (2014) Epigenetic responses to heat stress at different time scales and the involvement of small RNAs. Plant Signal. Behav. 9, e970430. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Tamura, K. , Peterson, D. , Peterson, N. , Stecher, G. , Nei, M. and Kumar, S. (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28, 2731–2739. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Tanabe, S. , Onodera, H. , Hara, N. , Ishii‐Minami, N. , Day, B. , Fujisawa, Y. , Hagio, T. , Toki, S. , Shibuya, N. , Nishizawa, Y. and Minami, E. (2015) The elicitor‐responsive gene for a GRAS family protein, CIGR2, suppresses cell death in rice inoculated with rice blast fungus via activation of a heat shock transcription factor, OsHsf23. Biosci. Biotechnol. Biochem. 80, 145–151. [DOI] [PubMed] [Google Scholar]
  56. Tang, Y. , Liu, Q. , Liu, Y. , Zhang, L. and Ding, W. (2017) Overexpression of NtPR‐Q up‐regulates multiple defense‐related genes in Nicotiana tabacum and enhances plant resistance to Ralstonia solanacearum . Front. Plant Sci. 8, 1963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Wang, C. , Zhang, Q. and Shou, H.X. (2009) Identification and expression analysis of OsHsfs in rice. J. Zhejiang Univ. Sci. B, 10, 291–300. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Wang, F. , Dong, Q. , Jiang, H. , Zhu, S. , Chen, B. and Xiang, Y. (2012) Genome‐wide analysis of the heat shock transcription factors in Populus trichocarpa and Medicago truncatula . Mol. Biol. Rep. 39, 1877–1886. [DOI] [PubMed] [Google Scholar]
  59. Wang, J. , Sun, N. , Deng, T. , Zhang, L. and Zuo, K. (2014) Genome‐wide cloning, identification, classification and functional analysis of cotton heat shock transcription factors in cotton (Gossypium hirsutum). BMC Genomics, 15, 961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Wang, Y. , Cai, S. , Yin, L. , Shi, K. , Xia, X. , Zhou, Y. , Yu, J. and Zhou, J. (2015) Tomato HsfA1a plays a critical role in plant drought tolerance by activating ATG genes and inducing autophagy. Autophagy, 11, 2033–2047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Wei, Y. , Shi, H. , Xia, Z. , Tie, W. , Ding, Z. , Yan, Y. , Wang, W. , Hu, W. and Li, K. (2016) Genome‐wide identification and expression analysis of the WRKY gene family in cassava. Front. Plant Sci. 7, 25. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Wei, Y. , Liu, G. , Bai, Y. , Xia, F. , He, C. and Shi, H. (2017a) Two transcriptional activators of N‐acetylserotonin O‐methyltransferase 2 and melatonin biosynthesis in cassava. J. Exp. Bot. 68, 4997–5006. [DOI] [PubMed] [Google Scholar]
  63. Wei, Y. , Liu, W. , Hu, W. , Liu, G. , Wu, C. , Liu, W. , Zeng, H. , He, C. and Shi, H. (2017b) Genome‐wide analysis of autophagy‐related genes (ATGs) in banana highlights MaATG8s in cell death and autophagy in immune response to Fusarium wilt. Plant Cell Rep. 36, 1237–1250. [DOI] [PubMed] [Google Scholar]
  64. Wiermer, M. , Feys, B.J. and Parker, J.E. (2005) Plant immunity: the EDS1 regulatory node. Curr. Opin. Plant Biol. 8, 383–389. [DOI] [PubMed] [Google Scholar]
  65. Wydra, K. , Banito, A. and Kpemoua, K.E. (2007) Characterization of resistance of cassava genotypes to bacterial blight by evaluation of leaf and systemic symptoms in relation to yield in different ecozones. Euphytica, 155, 337–348. [Google Scholar]
  66. Yabuta, Y. (2016) Functions of heat shock transcription factors involved in response to photooxidative stresses in Arabidopsis . Biosci. Biotechnol. Biochem. 20, 1–10. [DOI] [PubMed] [Google Scholar]
  67. Yamada, K. and Nishimura, M. (2008) Cytosolic heat shock protein 90 regulates heat shock transcription factor in Arabidopsis thaliana . Plant Signal. Behav. 3, 660–662. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Yoo, S.D. , Cho, Y.H. and Sheen, J. (2007) Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat. Protoc. 2, 1565–1572. [DOI] [PubMed] [Google Scholar]
  69. Yun, B.W. , Feechan, A. , Yin, M. , Saidi, N.B. , Le Bihan, T. , Yu, M. , Moore, J.W. , Kang, J.G. , Kwon, E. , Spoel, S.H. , Pallas, J.A. and Loake, G.J. (2011) S‐nitrosylation of NADPH oxidase regulates cell death in plant immunity. Nature, 478, 264–268. [DOI] [PubMed] [Google Scholar]
  70. Zhou, J. , Xu, X. , Cao, J. , Yin, L. , Xia, X. , Shi, K. , Zhou, Y. and Yu, J. (2018) HsfA1a is essential for Mi‐1.2‐mediated resistance to Meloidogyne incognita, and regulates Wfi1 transcription and H2O2 production. Plant Physiol, 176, 2456–2471. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

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Supplementary Materials

Additional Supporting Information may be found in the online version of this article at the publisher's website:

Fig. S1 The gene structure of 32 MeHsfs (Manihot esculenta heat stress transcription factors). The analysis of the phylogenetic relationship and gene structure of 32 MeHSFs using mega5.05 software and GSDS v2.0.

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Fig. S2 The conserved motif analysis of 32 MeHsfs (Manihot esculenta heat stress transcription factors). Link analysis of the phylogenetic relationship and conserved motifs of 32 MeHSFs using mega5.05 software and MEME v4.11.0.

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Fig. S3 The transcripts of 32 MeHsfs (Manihot esculenta heat stress transcription factors) in response to different treatments. For the gene expression assay, 30‐day‐old cassava leaves were sprayed with water (mock for flg22) or 10 μm flg22, or syringe infiltrated with 10 mm MgCl2 [mock for Xanthomonas axonopodis pv. manihotis (Xam)] or 108 colony‐forming units (cfu/mL) of Xam for 0, 1 and 6 h. Asterisks (*) indicate significant differences in comparison with mock treatment at P < 0.05.

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Fig. S4 The transcripts of MePRs (Manihot esculenta pathogen‐related genes) in gene‐silenced cassava leaves under mock conditions and pretreatment with salicylic acid (SA). Thirty‐day‐old cassava leaves were first syringe infiltrated with GV3101 strain containing the virus‐induced gene silencing (VIGS) vector for 14 days; thereafter, cassava leaves were sprayed with 10 µm salicylic acid (SA) and syringe infiltrated with 108 colony‐forming units (cfu)/mL of Xanthomonas axonopodis pv. manihotis (Xam). Different letters indicate significant differences in comparison with vector treatment at P < 0.05.

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Table S1 Identification of heat stress transcription factors (Hsfs), Enhanced Disease Susceptibility 1 (EDS1) and pathogen‐related genes (PRs) from cassava (Manihot esculenta).

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Table S2 The primers used in quantitative real‐time polymerase chain reaction.

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Table S3 The primers used in vector construction.

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