WANG Xiao-dong, BI Wei-shuai, GAO Jing, YU Xiu-mei, WANG Hai-yan, LIU Da-qun,

1 College of Plant Protection/Biological Control Center for Plant Diseases and Plant Pests of Hebei, Hebei Agricultural University,Baoding 071000, P.R.China

2 College of Life Sciences, Hebei Agricultural University, Baoding 071000, P.R.China

3 Graduate School of Chinese Academy of Agricultural Sciences, Beijing 100081, P.R.China

Abstract In Arabidopsis, systemic acquired resistance (SAR) is established beyond the initial infection by a pathogen or is directly induced by treatment with salicylic acid (SA) or its functional analogs, 2,6-dichloroisonicotinic acid (INA) and benzothiadiazole(BTH). NPR1 protein is considered the master regulator of SAR in both SA signal sensing and transduction. In wheat(Triticum aestivum) and barley (Hordeum vulgare), both pathogen infection and BTH treatment can induce broad-spectrum resistance to various diseases, including powdery mildew, leaf rust, Fusarium head blight, etc. However, three different types of SAR-like responses including acquired resistance (AR), systemic immunity (SI), and BTH-induced resistance (BIR)seem to be achieved by activating different gene pathways. Recent research on wheat and barley NPR1 homologs in AR and SI has provided the initial clue for understanding the mechanism of SAR in these two plant species. In this review,the specific features of AR, SI, and BIR in wheat and barley were summarized and compared with that of SAR in model plants of Arabidopsis and rice. Research updates on downstream genes of SAR, including pathogenesis-related (PR) and BTH-induced genes, were highlighted.

Keywords: systemic acquired resistance, NPR1, pathogenesis-related genes, wheat, barley

1. Introduction

Systemic acquired resistance (SAR) is an inducible form of plant defense that confers broad-spectrum immunity to secondary infections beyond the initial infection site.In Arabidopsis, SAR can be induced by either pathogen infection or treatment with salicylic acid (SA) or its functional analogs 2,6-dichloroisonicotinic acid (INA)and benzothiadiazole (BTH), which is associated with transcriptional activation of pathogenesis-related (PR)genes (Zheng and Dong 2013). The Arabidopsis NPR1 protein (Non-expresser of PR genes 1, also known as NIM1 and SAI1) is a master regulator required for SAR.Upon pathogen infection or SA/INA/BTH treatment, NPR1 translocates from the cytoplasm into the nucleus, where it interacts with the TGA2 transcription factor to promote the expression of multiple PR genes (Cao et al. 1994; Delaney et al. 1995; Ryals et al. 1997; Shah et al. 1997; Mou et al.2003). The endogenous SA level during plant defense response is largely dependent on the strength of cell death triggered by the pathogen (Fu and Dong 2013). The NPR1 paralogs NPR3 and NPR4 are involved in the degradation of NPR1 in a SA concentration-dependent manner (Fu et al.2012). At a low SA level, NPR1 is targeted for degradation in proteasomes by binding to NPR4. As the SA level increases after pathogen infection during basal resistance, SA binds to NPR4 and releases more NPR1, which activates the SAR.At an extremely high SA level during the hypersensitive response (HR), SA binds to NPR3 and promotes its interaction with NPR1, which finally leads to the turnover of NPR1 (Fu et al. 2012; Moreau et al. 2012). Interestingly, a bacterial Type III effector AvrPtoB was reported to mediate the degradation of NPR1 in the presence of SA via the host 26S proteasome (Chen et al. 2017).

In rice, a SAR-like enhanced resistance to Magnaporthe oryzae (Mo) can be induced by either BTH treatment or Pseudomonas fluorescens infection in a PR genes-involved manner (Smith and Métraux 1991; Schweizer et al. 1999;Oostendorp et al. 2001). In contrast to Arabidopsis, the endogenous level of SA in rice is relatively high and not elevated by pathogen infections (Silverman et al. 1995; Chen et al. 1997). Nevertheless, protein interactions between the rice homolog of NPR1 (NH1) and TGAs are conserved(Chern et al. 2001). The overexpression of Arabidopsis NPR1 (AtNPR1) in rice could enhance its resistance against multiple pathogens, including Xanthomonas oryzae pv.oryzae (Xoo), Mo, Fusarium verticillioides, and Erwinia chrysanthemi (Chern et al. 2001; Quilis et al. 2008; Xu et al.2017). Further studies indicate that the overexpression of NH1 leads to a higher level of resistance to Xoo and hypersensitivity to light and BTH (Chern et al. 2005; Bai et al. 2011). Interestingly, a WRKY transcription factor,OsWRKY45, was reported as another key regulator in the SA/BTH signaling pathway independent from NH1 in rice(Shimono et al. 2007; Nakayama et al. 2013).

Fig. 1 Models of systemic acquired resistance (SAR)-like responses in wheat and barley in comparison with SAR in Arabidopsis. In wheat and barley, both pathogen infection and benzothiadiazole (BTH) treatment can induce broad-spectrum resistance to various pathogens. Compared with SAR in Arabidopsis, three different types of SAR-like responses, including the acquired resistance(AR), systemic immunity (SI), and BTH-induced resistance (BIR), seem to be achieved by activating different gene pathways. Psj,Pseudomonas syringae pv. japonica; Mo, Magnaporthe oryzae; Xtc, Xanthomonas translucens pv. cerealis; SA, salicylic acid;MeJA, jasmonic acid methyl ester; ABA, abscisic acid.

However, SAR in wheat and barley is obviously different from those described above for Arabidopsis and rice (Fig. 1).In this review, the specific features of SAR in wheat and barley were summarized in comparison to SAR in model plants of Arabidopsis and rice. Our current knowledge on wheat/barley NPR1 homologs and downstream genes of SAR was highlighted.

2. SAR in wheat and barley

In wheat and barley, both the initial pathogen infection and BTH treatment can enhance plant resistance to secondary pathogen infection. However, different from SAR in Arabidopsis, three types of induced resistance have been reported in wheat and barley. These three biological processes show similarities with SAR, but they seem to be achieved by activating different gene pathways (Fig. 1).

Acquired resistance (AR) to secondary pathogen Mo can be induced in the area adjacent to the initial infection of Pseudomonas syringae pv. syringae DC3000 in a PR genesinvolved manner. Nevertheless, unlike SAR in Arabidopsis,this type of resistance is not systemic (Colebrook et al.2012). A previous study showed that the levels of both free and conjugated SA were significantly upregulated in barley leaves infiltrated with P. syringae but not in those infected with Blumeria graminis f. sp. hordei (Bgh) or Blumeria graminis f. sp. tritici (Bgt) (Vallélianbindschedler et al. 1998).Taken together, AR triggered by P. syringae DC3000 seems to be mediated by analogous pathways such as SAR in Arabidopsis and rice.

Systemic immunity (SI) in uninfected leaves of barley against secondary infection of Xanthomonas translucens pv. cerealis (Xtc) can be induced by Pseudomonas syringae pv. japonica (Psj) or Xtc (Dey et al. 2014). Different from SAR in Arabidopsis and rice, SI is associated with a local accumulation of abscisic acid (ABA), but not SA. It can also be induced by a local application of jasmonic acid methyl ester (MeJA) or ABA, but not SA or BTH. The same study also indicated that SI was not associated with NPR1, but with several WRKY and ERF transcription factors.

Several studies have demonstrated the presence of BTH-induced resistance (BIR) in wheat and barley to various pathogens, including Bgt and Puccinia triticina (Pt) (Gorlach et al. 1996; Hafez et al. 2014). However, most of the PR genes in wheat and barley were not induced by treatment with SA/INA/BTH (Vallélianbindschedler et al. 1998; Molina et al.1999). On the other hand, another group of BTH-induced genes, designated as wheat chemical-induced (WCI) and barley chemical-induced (BCI) genes, may be responsible for the BIR (Gorlach et al. 1996; Be?er et al. 2000).

3. Function of NPR1 in wheat and barley

Recent studies on wheat and barley NPR1 homologs have provided the initial clue to understand the signal sensing and transduction mechanisms of the previously described SAR-like responses. Wheat NPR1 homolog (wNPR1)showed protein interactions with four basic-region leucine zipper (bZIP) transcription factors (also known as TGAs),as well as two wheat orthologous copies of the rice NRR(Negative Regulator of Resistance) protein (Cantu et al.2013). Protein interactions between NPR1 and TGAs are critical for the functioning of NPR1 in Arabidopsis and rice(Chern et al. 2001; Després et al. 2003). In one of our recent studies, inductions of several barley PR genes,including HvPR1b, HvPR2, HvPR3_Chit2b, and HvPR5,were closely associated with the expression level of NPR1 in barley transgenic lines overexpressing wheat NPR1(wNPR1-OE) or suppressing barley NPR1 (HvNPR1-RNAi)during AR triggered by P. syringae DC3000 (Wang et al.2016). In the same study, a virulent effector protein from Puccinia striiformis f. sp. tritici (Pst) directly targeting wheat wNPR1 protein was discovered by a yeast two-hybrid screening assay. The NPR1-mediated PR gene inductions were suppressed by this effector possibly via its competitive effect on the protein interaction between NPR1 and TGA2.Interestingly, wheat transgenic lines overexpressing AtNPR1 or Secale cereal NPR1 (ScNPR1) showed enhanced resistance to Fusarium head blight (Makandar et al. 2006;Gao et al. 2013; Yu et al. 2017).

4. PR and BTH-induced genes in wheat and barley

We have summarized our current knowledge on the possible downstream genes of SAR in wheat and barley, including all the cloned PR and BTH-induced genes in Table 1. A neighbor-joining polygenetic tree was generated by MEGA Software using sequences of corresponding proteins aligned by MUSCLE method (Fig. 2).

So far, a total of 18 gene families from plant species were designated as PR genes in response to various pathogens(Loon et al. 2006; Ferreira et al. 2007; Sels et al. 2008).

PR1:Two genes in this gene family (PR1a and PR1b)have been cloned from both wheat and barley. PR1 showed induction by various pathogens (Gorlach et al.1996; Vallélianbindschedler et al. 1998; Molina et al. 1999;Gao et al. 2015) and was also reported as downstream of NPR1 in AR triggered by P. syringae DC3000 (Wang et al.2016). The expression level of wheat TaPR1b was much higher than that of TaPR1a in different tissues (Molina et al.1999). PR1 became sensitive to BTH treatment only in a wheat transgenic line overexpressing AtNPR1 (Molina et al.1999; Makandar et al. 2006), which indicates the presence of different signal sensing mechanisms between wNPR1 and AtNPR1. PR1 protein interacted physically with ToxA and potentially mediated ToxA-induced necrosis in sensitive wheat (Lu et al. 2014). A recent study provided genetic and biochemical evidence for the capacity of PR1 protein to bind sterols and inhibit pathogen growth (Gamir et al. 2017).

PR2:This gene family encodes β-1,3-glucanase and has been characterized in detail using immunolocalization method during the infection of wheat by Pst, Puccinia graminis tritici (Pgt), and Fusarium culmorum (Fc) (Sock et al. 1990; Kang and Buchenauer 2002; Liu et al. 2010).Barley HvPR2 showed clear involvement in NPR1-mediated AR triggered by P. syringae DC3000 (Wang et al. 2016).Wheat TaPR2 was also reported as a potential target of a virulent factor microRNA-like RNA 1 (Pst-milR1) from Pst(Wang et al. 2017).

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PR3, PR8, and PR11 (chitinase):Different types of chitinases were classified into three gene families as PR3(Type I, II, IV, VI, and VII), PR8 (Type III), and PR11 (Type V).Currently one chitinase from wheat (TaChit2) and three chitinases from barley (HvChit2a, HvChit2b, and HvChit2) were designated as PR3 (Gregersen et al.1997; Vallélianbindschedler et al. 1998;Gao et al. 2013). Transgenic tomato plants overexpressing wheat TaPR3_Chit2 showed enhanced resistance to F. oxysporum f. sp. lycopersici (Girhepuje and Shinde 2010). In addition, barley HvPR3_Chit2a has been validated as a downstream gene of NPR1 during AR triggered by P. syringae DC3000(Wang et al. 2016). Although PR8 and PR11 have not yet been designated in wheat or barley, several wheat/barley chitinases have shown involvement in the resistance to various pathogens.For instance, TaChit4 and HvChit26 were reported to be involved in wheat/barley resistance to F. graminearum(Fg) (Li et al. 2001; Shin et al. 2008).Induction of TaChitIb was discovered in wheat seedlings infected with Pgt (Yu et al. 1994). A purified barley chitinase expressed by Escherichia coli exerted antifungal activity against Alternaria alternate, F. oxysporium, and F. solani(Elhamid et al. 2010). In the polygenetic tree constructed in the review, we found that AtPR3 from Arabidopsis was closely clustered with HvChit26 and TaChitIb, but not with the designated wheat/barley PR3 proteins (Fig. 2).

PR4:This gene family encodes a chitin-binding protein and is reported to be sensitive to BTH treatment (Bertini et al.2003). The recombinant wheat TaPR4 protein expressed by E. coli inhibited the growth of Fc in vitro (Caruso et al. 2001).Barley HvPR4b also showed induction in AR triggered by P. syringae DC3000(Wang et al. 2016). Interestingly, a recent study in pepper (Capsicum annuum)indicated that PR4b was a cell death inducer and interacted with a leucine-rich repeat (LRR) protein (Sun et al. 2014).

Fig. 2 Polygenetic tree of pathogenesis-related (PR), barley chemical-induced (BCI), and wheat chemical-induced (WCI) proteins.Asterisk (＊) indicates expressed sequence tag (EST) or nucleotide accessions.

PR5:Genes encoding thaumatin-like proteins (TLP) are designated as PR5.This gene family showed induction by various pathogens in wheat and barley(Vallélianbindschedler et al. 1998; Reiss and Horstmann 2001; Wang et al. 2010;Li et al. 2015). A barley HvPR5_TLP6 has been confirmed as downstream of NPR1 during AR triggered by P. syringae DC3000 (Wang et al. 2016). Interestingly, wheat transgenic lines overexpressing or co-expressing TaPR2, TaPR3 and TaPR5 showed moderate resistance to Fg in both greenhouse and the field (Anand et al. 2003). In the polygenetic tree, wheat TaPR5 was clustered with barley HvPR5_TLP1, HvPR5_TLP2 and HvPR5_TLP4, whereas HvPR5_TLP5, HvPR5_TLP6, HvPR5_TLP7, and HvPR5_TLP8 were clustered together with PR5 proteins from Arabidopsis and Brachypodium (Fig. 2).

PR6:This gene family encodes a proteinase-inhibitor and has been cloned in both wheat and barley (Gaddour et al. 2001; Gao et al. 2013). Barley HvPR6 gene showed induction by several abiotic stimuli (Gaddour et al. 2001).The gene expression of wheat TaPR6 was slightly induced by F. asiaticum in a wheat transgenic line overexpressing AtNPR1 at the spike stage (Gao et al. 2013).

PR7:Genes encoding subtilisin-like proteinase from tomato were originally designated as PR7 (Jordá et al.2000). This gene family has not been cloned yet from wheat or barley. We have found the closest homolog of tomato SlPR7e and SlPR7f in barley from GenBank and temporarily designated as HvPR7 in our polygenetic analysis.

PR9:This gene family encodes peroxidase and was reported to be induced by various pathogens in wheat and barley including Bgt, Bgh and P. syringae(Thordal-Christensen et al. 1992; Gregersen et al. 1997;Vallélianbindschedler et al. 1998).

PR10:A gene family encoding ribonuclease-like protein was designated as PR10. A previous study revealed that barley HvPR10 was induced by Rhynchosporium secalis(Rs) (Steinerlange et al. 2003).

PR12:A group of small cysteine-rich antifungal proteins(AFP) from radish were originally designated as PR12(Terras et al. 1995). Although PR12 homologs in wheat or barley have not been cloned yet, a wheat transgenic line overexpressing radish RsPR12 showed enhanced resistance to Rhizoctonia cerealis without changing any agronomic traits (Lu et al. 2009). The closest homolog of radish RsPR12 in barley in the GenBank database was temporarily designated as HvPR12 in the polygenetic tree.

PR13:This gene family encodes thionins and was first designated in Arabidopsis (Epple et al. 1995). The closest homolog of Arabidopsis AtPR13 in barley in the GenBank database was temporarily designated as HvPR13 in the polygenetic tree.

PR14:A Bgh-induced barley lipid-transfer protein gene HvLTP4 was first designated as PR14 (Molina and García-Olmedo 1993; Garcíaolmedo et al. 1995). The expression levels of HvLTP2, HvLTP3, and HvLTP4 were induced by Bgh, but not by SA or INA (Molina and García-Olmedo 1993). Arabidopsis and tobacco transgenic lines overexpressing barley HvLTP2 showed enhanced tolerance to bacterial pathogens (Molina and García-Olmedo 1997).In another study, wheat TaLTP5 was used as a transgenic resource to improve wheat resistance to Cochliobolus sativus and Fg (Zhu et al. 2012). In contrast, another wheat LTP gene TaDIR1-2 was reported as a negative regulator in wheat resistance against Pst (Ahmed et al. 2017). A purified wheat lipid transfer protein expressed by E. coli exhibited a broad-spectrum antifungal activity against Alternaria sp., Rhizoctonia solani, Curvularia lunata, Bipolaris oryzae, Cylindrocladium scoparium, Botrytis cinerea, and Sarocladium oryzae (Kirubakaran et al. 2008).

PR15 and PR16:These two gene families encode oxalate oxidase and oxalate oxidase-like proteins,respectively, which are essential for the H2O2production during the HR triggered by Bgh in barley (Gregersen et al.1997; Zhou et al. 1998).

PR17:Members of this gene family have been cloned in barley and showed induction by Bgh (Christensen et al.2002). A recent study indicated that both barley HvPR1 and HvPR17c might be potential targets of a Bgh effector candidate CSEP0055. Further characterization of HvPR17c revealed that it was localized on the Bgh-induced papillae and exerted important functions in the defense response of barley (Zhang et al. 2012).

PR18:This gene family encodes fungus- and SA-inducible carbohydrate oxidases and was first discovered in sunflower (Custers et al. 2004). So far, the name of “PR18”has not been widely used (Loon et al. 2006). The closest homolog of the sunflower carbohydrate oxidases in barley from GenBank was temporarily designated as HvPR18 in the polygenetic tree.

BCI and WCI:A total of nine barley BCI and five wheat WCI genes were designated in previous investigations using BTH to enhance plant resistance to various pathogens(Table 1) (Gorlach et al. 1996; Be?er et al. 2000). Most of the barley BCI genes showed upregulation by BTH treatment (downregulation for HvBCI8), but none of them was sensitive to Bgh infection (Be?er et al. 2000). Whereas,all of the wheat WCI genes showed upregulation by both BTH treatment and Bgt infection (Gorlach et al. 1996). We have noticed some interesting relationships among PR, BCI,and WCI proteins in our polygenetic analysis (Fig. 2). For instance, both HvBCI1 and TaWCI2 are lipoxygenase and are clustered closely. For thionins or thionin-like proteins,HvPR13 and HvBCI2 are clustered closely. Moreover,TaWCI5 is clustered closely with PR17, especially with HvPR17a.

5. Conclusion

SAR-like responses observed in wheat and barley (AR, SI and BIR) show novel differences from SAR in Arabidopsis and rice. Recent studies on wheat and barley NPR1 homologs have provided the initial clue to understand the molecular mechanism behind these phenomena.Functions of key regulators in SAR, including NPR3,NPR4, and WRKY transcription factors, in wheat and barley require further exploration. More studies should be carried out to broaden our knowledge on the NPR1-mediated AR in the adjacent region of P. syringae DC3000 infection, WRKY and ERF transcription factors-mediated SI in uninfected leaves triggered by Psj or Xtc, as well as the molecular mechanism controlling BIR. Downstream genes involved in these biological processes, especially PR genes, have shown a great potential for use as transgenic resources for the improvement of the broad-spectrum resistance of wheat and barley to various pathogens. More efforts might need to be put in cloning and characterizing novel PR genes.It would be rather difficult but meaningful to explore the network of gene pathways among these different SAR-like responses.

Acknowledgements

We would like to acknowledge the National Natural Science Foundation of China (31701776, 31301649) and the Young Talents Project of Hebei Education Department, China(BJ2016028).