Background

Plants are always attacked by diverse and widespread potential pathogens, which cause numerous diseases. These diseases lead to 16% of global crop yield losses (Oerke 2006). Plants have evolved sophisticated innate ability of each cell to fend off the attack (Spoel and Dong 2012). There are two-layered system involved in plant immune response. The first layer is governed by cell surface-localized pattern recognition receptors (PRRs) that detect pathogen-associated molecular patterns (PAMPs), such as bacterial flagellin or fungal chitin, which are highly conserved molecules essential for the pathogen’s life cycle, and trigger a relatively weak immunity (PTI). PTI comprises a wide array of responses, including the production of reactive oxygen species (ROS), increases in intracellular calcium concentration, callose deposition in cell wall, antimicrobial compounds called phytoalexins and the activation of mitogen-activated protein kinases (MAPKs) (Leach et al. 2014). It is a broad-spectrum resistance that wards off most invading organisms. To counter PTI, the pathogens evolved mechanisms to secret and deliver highly variable effectors into host cells to suppress PTI, which is called effector-triggered susceptibility (ETS). The second layer of plant defense acts largely inside the cell and is based on highly polymorphic resistance proteins which directly or indirectly recognize specific virulence effectors secreted within host cells by pathogens, inducing the effector-triggered immunity (ETI). ETI is a rapid and stronger resistance response, usually associated with programmed cell death at sites of infection, termed the hypersensitive response (HR). Other defense responses include the production of ROS, enhancement of cell walls, accumulation of toxic metabolites or proteins, and altered levels of hormone (Leach et al. 2014).

The ancient domesticated crop, rice (Oryza sativa L.) is the most important staple food for humans and is one of the most widely cultivated crops all over the world (Ainsworth 2008). Though rice production has been almost doubled over the recent decades due to the introduction of the semi-dwarf gene sd1, hybrids, and improvements in cultivation management practices, it needs to significantly increase in order to meet the projected demand from the ever-expanding human population (Khush 2005; Skamnioti and Gurr 2009). However, the increase is challenged by farmland availability, water, soil fertility, climate change, insects and diseases. Rice is vulnerable to a number of diseases caused by bacteria, viruses, or fungi (Dai et al. 2000; ** sub-CSSLs in rice. PLoS One 9:e95751" href="/article/10.1186/s12284-019-0358-y#ref-CR96" id="ref-link-section-d53682158e717">2014).

Though Xoc and Xoo are highly related bacterial species, they infect rice in different ways. Xoo enters leaf through the hydathodes or wounds, multiplies in the intercellular spaces of the underlying epitheme, and propagate to reach the xylem vessels. The bacteria move through the veins of leaves and spread into the plant. Water-soaked spots at the leaf tips and margins were first observed. Then, the leaves become chlorotic and necrotic along the leaf veins (Lee et al. 2011) (Fig. 1a). Xoc penetrates the leaf mainly through stomata or wounds, multiplies in the substomatal cavity and then colonizes the intercellular spaces of the parenchyma. Different from BB, small, water-soaked lesions anywhere along the leaf between the veins were observed during the early stage of BLS infection, resulting in translucent and yellow streaks (Fig. 1b). The infected leaves turn greyish white and die later on (Nino-Liu et al. 2006).

Fig. 1
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Symptoms of (a) bacterial light caused by Xanthomonas oryzae pv. oryzae and (b) bacterial leaf streak caused by Xanthomonas oryzae pv. oryzicola

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Diverse effector proteins with virulence, avirulence functions or both are secreted by Xanthomonas oryzae. Among them, transcription activator like (TAL) effector proteins are a structurally and functionally distinct class of proteins secreted into plant cells by a type III secretion (T3S) system. TAL effectors (also termed as TALEs) import in the nucleus and bind to TALE-specific DNA, which is termed as effector binding elements (EBEs). The recognition transcriptionally activates host target genes, resulting in susceptibility or resistance (Bogdanove et al. 2010; Bogdanove and Voytas 2011).

Disease Resistance Genes and the Interactions

Deployment of gene-conferred host plant resistance provides an economical, effective, environment friendly approach to control plant diseases and minimize the losses. Extensive genetic studies on rice resistance to BB have been conducted over the last 20 years. To date, more than 40 resistance (R) genes conferring host resistance to various strains of Xoo have been identified and 11 of them were cloned, namely Xa1, Xa3/Xa26, Xa4, xa5, Xa10, xa13, Xa21, Xa23, xa25, Xa27, and xa41 (Table 1) (Ji et al. 2018). These R genes can be classified into four groups based on their encoding proteins, including receptor-like kinase (RLK) genes (Xa21, Xa3/Xa26 and Xa4), sugar will eventually be exported transporter (SWEET) genes (xa13, xa25 and xa41), executor genes (Xa10, Xa23 and Xa27) and other types of genes (Xa1 and xa5). Some of these isolated R genes are widely employed in rice breeding programs to control BB, such as Xa3/Xa26 and Xa4, which played an important role in controlling the disease in Asia since 1970s. Nearly all the commercial indica hybrid rice varieties in China are known to contain Xa4, and Xa3/Xa26 is widely distributed in both indica and japonica varieties in China (Deng et al. 2018; Hu et al. 2017). The cognate avirulence (Avr) genes to all the R genes except Xa4 have been reported (Table 1).

Table 1 Summary of the cloned rice R genes and the cognate Xanthomonas oryzae Avr genes
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In contrast to BB, no native major R gene controlling resistance to BLS has been identified in rice and only a few of quantitative resistance loci have been mapped. Interestingly, one of them, qBlsr5a, with relatively large effect, was mainly controlled by xa5 (** sub-CSSLs in rice. PLoS One 9:e95751" href="/article/10.1186/s12284-019-0358-y#ref-CR96" id="ref-link-section-d53682158e1556">2014). A non-host R gene, Rxo1, was isolated from maize, and the transgenic rice with Rxo1 has been proved to confer high level resistance to BLS (Zhao et al. 2005).

In addition, some defense-related or susceptible genes in rice were reported to be involved in the interaction with Xoc (Shen et al. 2010; Tao et al. 2009). Here, we focus on the recent advances in identification of the R genes or their products and the cognate pathogen effectors. The underlying molecular mechanisms of the interaction between rice and Xoo or Xoc are discussed. Additionally, two genes, Xa7 and Xo1, which have not been cloned yet, are also discussed due to their potential value in rice breeding programmes and special features. To date, most of the cloned plant R genes encode nucleotide-binding and leucine-rich repeat domain (NLR) proteins (Li et al. 2015).

However, only one encodes NLR protein among the 11 cloned Xa genes (Yoshimura et al. 1998). These Xa genes are classified into four groups based on the encoded protein types including RLK (receptor-like kinase), SWEET (sugar will eventually be exported transporter), executor R proteins and other proteins.

Receptor-Like Kinase (RLK) Genes

In plants, PRRs, which can recognize diverse pathogen-associated molecular patterns are a key component of the innate immune system. All the known plant PRRs are either transmembrane receptor-like kinases (RLKs) or transmembrane receptor-like proteins (RLPs) (Antolín-Llovera et al. 2012). There are over 1100 candidate RLKs/RLPs in rice genome (Shiu et al. 2004). RLKs typically contain an extracellular domain, a single-pass transmembrane domain, and an intracellular kinase domains, whereas RLPs lack the kinase domain (Monaghan and Zipfel 2012). Leucine-rich repeat receptor-like kinases (LRR-RLKs) represent the largest subfamily of plant RLKs (Afzal et al. 2008).

The LRR-RLK gene Xa21, originated from the wild rice species Oryza longistaminata, was the first cloned R gene in rice (Song et al. 1995). Xa21 have been proved to confer broad-spectrum resistance to Xoo. However, Xa21-mediated resistance progressively increases from the susceptible juvenile two-leaf stage through later stages, with full resistance only at the adult stage (Century et al. 1999; Wang et al. 1996). Overexpression of Xa21 gene can enable plants with resistance at both seedling and adult stages (Park et al. 2010a). The regulation of Xa21-mediated immunity has been extensively and comprehensively studied. Several XA21 binding proteins (XBs) with diverse functions have been characterized (Table 2, Fig. 2). The phosphorylation state of XA21 is important for its function. In the absence of infection, the ATPase XB24 physically associates with the XA21 juxtamembrane domain and promote phosphorylation of specific serine and threonine residues to maintain the inactive state of the XA21 protein. On recognition of pathogen invasion, the XA21 kinase disassociates from XB24 and triggers downstream defense responses (Chen et al. 2010b). After activation, XB15, a PP2C phosphatase, acts on XA21 and dephosphorylates the autophosphorylated XA21 (Park et al. 2008). The Xoo tyrosine-sulfated and type I-secreted protein RaxX is the ligand to induce the XA21-mediated immunity (Pruitt et al. 2015). The sulfated RaxX directly binds XA21 with high affinity (Luu et al. 2019). More details are shown in Table 2 and Fig. 2.

Table 2 Summary of XA21-binding proteins
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Fig. 2
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Xa21-mediated immune signaling pathways triggered by Xanthomonas oryzae. Sulphated RaxX is recognized by XA21 and activate XA21-mediated resistance. Several XA21 binding proteins, including OsSERK2, XB3, XB10, XB15, XB21, XB24, XB25, Bip3 and SDF2 are involved in regulating XA21-mediated resistance. XA21 is processed in endoplasmic reticulum, which is negatively and positively regulated by the ER chaperones BiP3 and SDF2, respectively. OsSERK2 positively regulates the immunity by forming a constitutive complex with XA21 and transphosphorylating XA21. XB24 binds to XA21 and promotes autophosphorylation of XA21 to keep it in an inactive state. During Xoo infection, XB24 dissociates from XA21. XB3 and XB25 are required for XA21 accumulation. XB15 dephosphorylates the autophosphorylated XA21 and attenuates the XA21-mediated resistance. XB21 functions as an auxilin to positively regulate XA21-mediated immunity. The transcription factor XB10/OsWRKY62 acts as a negative regulator XA21-mediated immunity

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Another LRR-RLK gene Xa26, was originally identified from indica variety Minghui 63, an elite restorer line of hybrid rice in China (Sun et al. 2004). Further study demonstrated Xa3, identified in a japonica variety Wase Aaikoku 3, is the same gene as Xa26 (**ang et al. 2006). OsSERK2 and OsTPI1.1 interact with XA3/XA26 and are involved in XA3/XA26-mediated resistance (Chen et al. 2014; Liu et al. 2018). OsTPI1.1 encoding a triosephosphate isomerase (TPI) catalyzes the reversible interconversion of dihydroxyacetone phosphate to glyceraldehyde-3-phosphate. Reduced expression of OsTPI1.1 largely compromises XA3/XA26-mediated resistance. OsTPI1.1 participates in the defense response through TPI which is significantly enhanced by binding with XA3/XA26 (Liu et al. 2018). As well as XA21, XA3/XA26-mediated resistance is positively regulated by OsSERK2 (Chen et al. 2014). AvrXa3, the cognate avirulence gene to XA3/Xa26, has been isolated, but how it initiates XA3/XA26-meditaed resistance remains unclear (Li et al. 2004).

Xa4, encoding a cell wall-associated kinase, confers a race-specific resistance to Xoo at all stages of rice growth (Leach et al. 2001; Sun et al. 2003; Hu et al. 2017). Wall-associated kinases (WAKs) are also a subfamily of RLKs that physically link the cell wall with the plasma membrane to transmit extracellular signals to the cytoplasm (Anderson et al. 2001). Xa4 was first introgressed into commercial rice varieties in the early 1970s. It is one of the most widely employed resistance genes in breeding programs. Nearly all the indica hybrid rice cultivars in China carry Xa4 (Leach et al. 2001). XA4 prevents the invasion of Xoo through reinforcing the cell wall (Hu et al. 2017). The accumulation of the two phytoalexins, sakuranetin and momilactone A, which are likely to suppress Xoo in plant, is proved to be associated with Xa4-mediated resistance. In addition to conferring durable resistance to Xoo, Xa4 increases the mechanical strength of the culm and reduces the plant height slightly, and thus may enhance the lodging resistance (Hu et al. 2017). The multiple favorable agronomic traits related with Xa4 may explain why it is widely used.

Sugar Will Eventually be Exported Transporter (SWEET) Genes

Three recessive R genes, xa13, xa25 and xa41, encodes clade III SWEET proteins. SWEET, a unique family of sugar efflux transporters, play a vital role in various biological processes, including pollen nutrition, senescence, seed filling and plant-pathogen interactions (Chen et al. 2012; Guan et al. 2008; Quirino et al. 1999; Streubel et al. 2013). SWEETs are grouped into a four-clade phylogenetic tree in plants (Eom et al. 2015). There are 17 and 22 SWEET genes in Arabidopsis and rice genomes, respectively (Chen et al. 2010a). Over the last 10 years, several studies have suggested sugar exporting into the apoplast via clade III SWEETs is hijacked by TAL effectors of pathogen, which is essential for pathogen growth and virulence (Eom et al. 2015). The xa13 (also known as Os8N3 and OsSWEET11) confers specific resistance to Xoo race 6, which was originally identified in cultivar BJ1 (Chu et al. 2006). It was isolated through different strategies by two groups (Chu et al. 2006; Yang et al. 2006). The TAL effector PthXo1 from Xoo directly targets to the EBEs, in the promoter of dominant Xa13 but not xa13 alleles to induce its expression, which is critical for susceptibility (Fig. 3) (Römer et al. 2010; Yuan et al. 2009). Further studies showed that the XA13 protein cooperates with two copper transporters, COPT1 and COPT5, to participate in copper redistribution. Copper is widely used as an important element for pesticides in agriculture. XA13, COPT1 and COPT5 are employed by TAL effectors of Xoo and remove toxic Cu from xylem vessels, where pathogen multiplies and spreads to cause disease (Yuan et al. 2010). Interestingly, knock-out of OsSWEET11 showed increased resistance to Rhizoctonia solani, which causes sheath blight disease. It suggests that OsSWEET11 may also be employed by the fungal pathogen Rhizoctonia solani (Gao et al. 2018). In addition, Xa13 was found to be required for pollen development. The Xa13-silenced plants had low fertility, and most pollen grains were defective in comparison with normal pollen grains (Chu et al. 2006).

Fig. 3
figure 3

The SWEET gene xa13 and the executor gene Xa23 mediated immune signaling pathways triggered by Xanthomonas oryzae. The TALEs are secreted into the cytoplasm of plant cells through the type III secretion system, enter the nucleus, bind to the specific promoter elements and induce the expression of Xa13. XA13 is hijacked by TALEs to export sucrose to the apoplast, which provides nutrition to the pathogen. XA13 is also employed together with COPT1 and COPT5 by TALEs to remove toxic Cu from xylem vessels. The resistant allele xa13 with mutations in the EBEs disrupt the binding of TALEs, leading to disease resistance. Like SWEET genes, the executor R gene Xa23 is transcriptionally activated by TALEs, triggering host defense responses

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As well as Xa3/Xa26, xa25 (also known as OsSWEET13) was identified from Minghui 63 (Chen et al. 2002). It confers race-specific resistance to Xoo strain PXO339 at both seedling and adult stages. Similar to xa13, the expression of dominant Xa25 but not recessive xa25 was rapidly induced by PXO339 (Liu et al. 2008; Lee et al. 2003). AvrXa10 specifically induces Xa10 expression through direct binding Xa10 promoter. Rice plants with constitutive but weak expression of Xa10 showed lesion mimic phenotype. Further study has revealed that XA10 forms hexamers and locate in the ER membrane of plant and HeLa cells, which mediates the disruption of the ER, cellular Ca2+ homeostasis and triggers programmed cell death (Tian et al. 2014).

Another executor gene Xa23 isolated from a wild rice species of O. rufipogon, confers an extremely broad spectrum of resistance to Xoo strains isolated from different regions at all growth stages of rice. Similar to Xa27, Xa23 shares identical ORF with the susceptible xa23 allele, and a 7-bp polymorphism in the promoter regions leads to induction of Xa23, but not xa23, by AvrXa23. Transient expression analysis indicated that XA23 triggers HR in N. benthamiana and tomato (Wang et al. 2015). AvrXa23 was found to be highly conserved in all the tested Xoo isolates (Wang et al. 2014). It is possible that AvrXa23 contributes to the virulence of Xoo for infection or growth in host plants. The prevalence of AvrXa23 in natural Xoo strains explains why Xa23 shows the broad-spectrum resistance.

Other Genes

In rice genome, 480 nucleotide-binding domain and leucine-rich repeat (NLR) genes have been revealed, but only a single one, Xa1, conferring resistance to Xoo, was isolated (Yoshimura et al. 1998). Xa1 was isolated from japonica cultivar Kogyoku and its expression was induced by bacterial infection and wounding (Yoshimura et al. 1996). Xa1 confers resistance against Xoo by recognizing several TAL effectors including PthXo1, Tal4 and Tal9d, but truncated interfering TAL effectors (also termed as iTALEs). The iTALEs may function as decoys interfering with the recognition of intact TALEs by XA1 and block its function (Ji et al. 2016a).

The recessive gene xa5 confers broad resistance spectrum to Xoo and is most commonly found in the Aus-Boro varieties from Bangladesh. The xa5 is a natural allele of Xa5 for the transcription factor IIA gamma subunit 5 (TFIIAγ5), contains a mutation in the 39th residue, in which the valine (V) residue is replaced with glutamine (E) (V39E) (Jiang et al. 2014).

The non-host resistance gene, Rxo1 encoding a NLR protein, confers high level resistance to Xoc in rice. It also controls resistance to the pathogen Burkholderia andropogonis, which causes bacterial stripe of sorghum and maize. Transgenic lines with Rxo1 also showed HR when inoculated with avrRxo1 containing Xoc strain (Zhao et al. 2004a; Zhao et al. 2004b; Zhou et al. 2008). The durable resistance of Xa7 is due to a fitness penalty in Xoo associated with adaptation to Xa7 (Vera Cruz et al. 2000; Bai et al. 2000). Mutations occurred specifically at the avrXa7 gene in the adapted strains, which displayed reduced aggressiveness on susceptible rice cultivars (Vera Cruz et al. 2000; Bai et al. 2000). Additionally, Xa7 are more effective at high temperatures, whereas other R genes are less effective (Webb et al. 2010). Another yet uncharacterized gene Xo1, was identified in the American heirloom rice variety Carolina Gold Select, and confers resistance to the tested African strains of Xoc, but not Asian strains (Triplett et al. 2016). Like Xa1, Xo1-mediated recognition of full-length TALEs can also be blocked by truncated TALEs (Read et al. 2016). Interestingly, Xa1 and Xo1 are located in the same region (Triplett et al. 2016). Further studies are needed to determine whether Xo1 is controlled by Xa1 or another gene.

Breeding Strategies to Develop Broad-Spectrum and Durable Resistance to Xoo and Xoc

Use of host plant resistance is generally the most favorable tactic to control diseases due to economic and environmental reasons. Marker-assisted selection (MAS) and genetic transformation are the two major approaches for R gene deployment in plant breeding programs. However, controversy on food safety and constraints on regulatory in some countries have serious plagued the application of genetically modified varieties. MAS, free of political issues and social problems, is more widely used by breeders. Pyramiding R genes resistant to different races of the pathogen through marker-assisted breeding strategies, is a very effective way to achieve durable and broad-spectrum resistance, while employment of a single R gene and adaption of the pathogen often lead to resistance breakdown in a short period.

Based on the previous reports, xa5, Xa7, xa13, Xa21 and Xa23 are more frequently used by rice breeders due to the comparatively broader spectra of resistance. Xu et al. (2012) transferred Xa7 and Xa21 into Yihui 1577, an elite hybrid rice restorer line. The pyramiding lines and their derived hybrids displayed resistance to all the seven Xoo strains, while the lines containing single Xa7 or Xa21 were resistant to six of them. Two Basmati rice varieties PB1121 and PB6 were improved for resistance to BB (xa13 and Xa21) through MAS (Ellur et al. 2016). In another study, three genes, xa5, xa13 and Xa21 were transferred into Lalat, a popular indica variety in Eastern India but susceptible to bacterial blight (Dokku et al. 2013). The improved lines showed significant enhanced resistance.

Because Xa23 displays broadest resistance, it is often used alone, or along with R genes against rice blast disease or/and brown planthopper (Zhou et al. 2011; Huang et al. 2012; Ni et al. 2015; Jiang et al. 2015; Ji et al. 2016b; ** of previously identified resistance loci with large effect, including the dominant locus Xo1 and the recessive locus bls1, will facilitate employment of them in rice breeding programmes (Triplett et al. 2016; He et al. 2012).

In our breeding practice, we introgressed Xa7 + Xa21 into an elite restorer line R900 of hybrid rice through marker-assisted backcrossing (MABC) scheme in less than 3 years, which is much more efficient than the conventional breeding method. The improved lines recovered more than 99% genome background of the recurrent parent R900, and showed a broad-spectrum resistance to Xoo without any significant difference in main agronomic traits in both the growth chambers and paddy fields (unpublished data). In addition, the R genes can be used separately in time and space. Development of near-isogenic lines and rotation of the R genes could reduce the selection pressure on pathogens and maximize the life span of R genes. Multi-lines containing different R genes also has the potential to provide broad-spectrum and durable disease resistance.

In recent years, the emerging genome-editing technologies, including zinc-finger nucleases (ZFNs), TAL effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 (CRISPR-associated protein-9 nuclease), have revolutionized biology by enabling targeted modifications of genomes (Christian et al. 2010; **ek et al. 2012; Kim et al. 1996). These technologies have been successfully applied in model species Arabidopsis thaliana, Nicotiana benthamiana and multiple crops including rice, wheat, maize, barley, soyben, tomato, potato, citrus, and sorghum (Shah et al. 2018). The powerful tools have great potential in improving the plant disease resistance. Elimination of EBEs in promoters of susceptibility genes or adding EBEs to promoters of executor R genes through genome editing, could enhance the resistance to BLB. For example, the EBEs of AvrXa7 and PthXo3 in the OsSWEET14 promoter were precisely edited by TALENs, which prevents the induction by TALEs. The mutated lines showed strong resistance to both AvrXa7- and PthXo3-dependent Xoo strains (Li et al. 2012). Similarly, the promoter of Xa13 (OsSWEET11) was targeted by CRISPR/Cas9-based disruption, leading to enhanced resistance without affecting rice fertility (Li et al. 2019). In a very recent study, EBEs in the promoters of OsSWEET11, OsSWEET13 and OsSWEET14 were edited simultaneously by CRISPR/Cas9 technology and rice lines conferring broad-spectrum resistance to Xoo were created (Xu et al. 2019). In another study, six EBEs corresponding three TALEs from Xoo and three from Xoc, were added to the Xa27 promoter, resulting in broad-spectrum resistance to both Xoo and Xoc (Hummel et al. 2012). It suggests that engineering of EBEs upstream of rice executor R genes through genome-editing technologies is a potential strategy to generate germplasms with broad-spectrum resistance to Xoo, Xoc and other bacterial pathogens.

Conclusions

Rice-Xanthomonas oryzae patho-system is a powerful model for research toward solutions in disease control. Although tremendous progress has been made in the past decades, there are still many queries and challenges. For example, whether there is any major R gene in rice against BLS? The xa5 confers resistance to both Xoo and Xoc, does any other identified Xa genes have the same effect? The ligand from Xoo mediating Xa4 resistance is still not determined. The partners and/or components associated with R proteins remain largely unknown in rice. It will be interesting to understand how R genes activate downstream signaling components and trigger plant defense response system. TAL effectors injected into plant cells have to be translocated into nucleus to bind to the target S or R genes for virulence or plant immunity. However, the underlying mechanism needs to be further elucidated. Altogether, a comprehensive understanding of the molecular interactions between rice and Xanthomonas oryzae is the pivotal for more efficient and durable disease control.