TuRLK1, a leucine-rich repeat receptor-like kinase, is indispensable for stripe rust resistance of YrU1 and confers broad resistance to multiple pathogens

YrU1 is a nucleotide-binding site (NBS) and leucine-rich repeat (LRR) protein (NLR), with additional ankyrin-repeat and WRKY domains and confers effective resistance to stripe rust fungus Puccinia striiformis f. sp. Tritici (Pst). YrU1 was positionally cloned in the progenitor species of the A genome of bread wheat, Tricicum urartu, recently. However, the molecular mechanism and components involved in YrU1-mediated resistance are not clear. In this study, we found that the transcript level of TuRLK1, which encodes a novel leucine-rich repeat receptor-like kinase, was up-regulated after inoculation with Pst in the presence of YrU1, through RNA-seq analysis in T. urartu accession PI428309. TuRLK1 contained only a small number of LRR motifs, and was localized in the plasma-membrane. Transient expression of TuRLK1 induced hypersensitive cell death response in N. benthamiana leaves. Silencing of TuRLK1, using barley stripe mosaic virus (BSMV)-induced gene silencing (VIGS) system in PI428309 that contains YrU1, compromised the resistance against stripe rust caused by Pst CY33, indicating that TuRLK1 was required for YrU1-activated plant immunity. Furthermore, overexpression of TuRLK1 could enhance powdery mildew resistance in bread wheat and Arabidopsis thaliana after inoculating with the corresponding pathogens. Our study indicates that TuRLK1 is required for immune response mediated by the unique NLR protein YrU1, and likely plays an important role in disease resistance to other pathogens.

of NLRs) signaling plays a key role in PTI, and TIR signaling mutants exhibit attenuated PTI responses and decreased resistance against Pto DC3000 hrcC and Hyaloperonospora arabidopsidis (Hpa) Noco2 in Arabidopsis thaliana (hereafter, Arabidopsis) [5]. PTI induced by Pto DC3000 hrcC, flg22 and nlp20 in Arabidopsis requires components of ETI, such as signaling-competent dimers of the lipase-like proteins EDS1 and PAD4, ADR1 family helper NLRs [5,6]. Moreover, in Arabidopsis, ETI activated by NLRs could increase the production of ROS and the expression of PTI-responsive genes induced by PAMPs to enhance PTI defense responses [7]. Nevertheless, on the other hand, potentiation of PTI is an indispensable component of ETI during bacterial infection in Arabidopsis, supported by substantial evidence [7,8]. For example, PRR and its co-receptors Arabidopsis mutants, fls2 efr cerk1and bak1 bkk1 cerk1, could not show an effective ETI response against Pst DC3000 (avrRpt2, AvrPphB or AvrRps4) [8]. Similarly, two other mutants, bak1-5 bkk1-1 and fls2 efr, both showed a higher susceptibility to Pst DC3000 (AvrRps4) than wild-type plants [7]. Notably, PTI was demonstrated being able to potentiate ETI-induced cell death. Activation of PTI or ETI AvrRpp4/ AvrRps4 alone could not result in cell death, except for coactivation of PTI and ETI AvrRpp4/AvrRps4 . Those findings indicate that ETI and PTI mutually potentiate and interdepend with each other. However, those studies are performed only in model plants Arabidopsis with bacterial pathogens.
PRRs mainly consist of receptor-like kinases (RLKs) and receptor-like proteins (RLPs). In plants, RLKs are a large family of proteins, which contain an ectodomain (ECD), a single pass transmembrane domain, and a cytoplasmic kinase domain, while RLPs lack the intracellular kinase domain [9,10]. The ECDs of RLKs are highly variable, including leucine-rich repeat (LRR) domain, lysine motifs (LysM), lectin domain, or epidermal growth factor (EGF)-like domain, providing means to recognize a wide range of ligands, such as steroids, peptides, polysaccharides, and lipopolysaccharides [9]. Notably, members of the LRR-RLK subfamily contain varied numbers of LRRs, and the role of the LRR-RLK is often dependent on the number of LRRs in the PTI. In Arabidopsis, LRR-RLKs with large numbers of LRRs often function as PRRs to perceive PAMPs or danger signals, such as FLS2, EFR, and PEPR1/2, which contain more than 20 LRRs. In contrast, LRR-RLK proteins with small numbers of LRRs often function as co-receptors for PRRs, such as BAK1 and SOBIR1, or regulatory proteins in the immune complex, such as BIR1 and BIR2, which contain only 4 to 6 LRRs. Stripe rust, caused by biotrophic pathogen Pst, is one of the most devastating diseases of wheat (Triticum aestivum L.) and severely reduces bread wheat yields around the world [11][12][13]. To date, although over 80 stripe rust resistance loci have been identified and mapped in Triticum spp., only nine genes, Yr5, Yr7, YrSP, Yr15, Yr18/Lr34, Yr36, Yr46, YrAS2388 and YrU1, have been cloned [14][15][16][17][18][19][20][21][22]. Among them, YrAS2388 encodes a typical coiled-coil (CC) NLR, and Yr5, Yr7 and YrSP all encode NLRs with a non-canonical N-terminal zincfinger BED domain [19]. YrU1 encodes an NLR with an N-terminal ankyrin-repeat (ANK) domain and a C-terminal WRKY domain, which was cloned in Triticum urartu, the progenitor species of the A genome of bread wheat. The NLRs with ANK and WRKY domains are rare, and only exist in the genomes of wheat and its relatives [15]. YrU1 likely functions as a typical NLR that elicits effective ETI after recognition of the cognate effector proteins derived from biotrophic pathogen Pst. How YrU1 activates plant immunity, and whether pattern-recognition receptors/co-receptor or other key components of PTI are required for YrU1-mediated plant immunity are remained to be determined.
In the RNA-seq data of T. urartu accession PI428309, which contains the functional YrU1 gene, we found the expression of a novel leucine-rich repeat receptor-like kinase gene, designated TuRLK1, was induced after infection of Pst CYR33. TuRLK1 contains six LRR motifs, localizes in the plasma-membrane. Overexpression of TuRLK1 could induce strong cell death in N. benthamiana leaves, suggesting that TuRLK1 may play an important role in plant immune response. Silencing of TuRLK1, using barley stripe mosaic virus (BSMV)-induced gene silencing (VIGS) system in PI428309, severely compromised the stripe rust resistance of YrU1 against Pst CY33, indicating that TuRLK1 was required for YrU1-mediated plant immunity. This study provides evidence that an RLK, the key PTI component, is indispensable for ETI, in the fungal disease resistance, which is in consistent with the previous studies on bacterial disease resistance. Furthermore, over-expression of TuRLK1 could enhance the powdery mildew resistance in bread wheat cultivar Fielder and Arabidopsis thaliana accession Col-0, which indicates the potential role of TuRLK1 in resistance breeding against multiple disease pathogens.

Result
The expression of TuRLK1 was up-regulated after inoculation with Pst CY33 in Triticum urartu PI428309 In order to identify important PTI components involved in YrU1-mediated stripe resistance, RNA-seq data was analyzed in T. urartu accession PI428309 [23], which has the functional YrU1 gene. The transcripts of a novel leucine-rich repeat receptor-like kinase gene, designated TuRLK1, were increased significantly after Pst CYR33 infection in PI428309 (Fig. 1a). We verified the results of RNA-seq via examining the transcript levels of TuRLK1 at 0 h post inoculation (hpi), 12 hpi, 24 hpi and 36 hpi with Pst CYR33 by qRT-PCR. The accumulation of transcripts of TuRLK1 was at much higher level at 12 hpi, 24 hpi and 36 hpi as shown in Fig. 1b. These results implied that TuRLK1 may be involved in the resistance to wheat stripe rust conferred by YrU1.
The coding sequence of TuRLK1 in PI428309 encodes a protein of 660 amino acids, and the relative molecular weight of TuRLK1 is about 73 kDa. TuRLK1, with an N-terminal LRR domain, a transmembrane region and a C-terminal Ser/Thr/Tyr Kinase domain, has the typical structure of RLK family members (Fig. 1c). TRIUR3_02522, the TuRLK1 in the susceptible T. urartu accession G1812, has 666 amino acids, which is nearly identical to TuRLK1 in PI428309, except for the number of LRR motifs. TuRLK1 in PI428309 has six LRR motifs, with two more LRR motifs than TRIUR3_02522 (Fig. 1c). As shown in Fig. 1d, the protein sequence of TuRLK1 in PI428309 is greatly different from the well-known RLKs in Arabidopsis thaliana. To identify the allelic variants of TuRLK1 in the Triticeae, we searched protein databases and conducted a multiple sequence alignment with TuRLK1 and 4 homologous proteins of TuRLK1 from different Triticeae accessions. The alleles identified from different Triticeae accessions are almost identical, except for a few amino acids (Additional file 1), indicating that TuRLK1 is well conserved in the Triticeae.

TuRLK1 is localized in plasma membrane
For examining the localization of TuRLK1, we created two constructs expressing TuRLK1 and Green/Yellow Fluorescent Protein (GFP/YFP) fusion proteins, respectively. After transiently expressing them in Nicotiana benthamiana and wheat leaves, TuRLK1 proteins were observed mainly localized in plasma membrane of both N. benthamiana and wheat cells using confocal microscope ( Fig. 2a and b). This result is consistent with the localization characteristics of most reported RLKs.

Overexpression of TuRLK1 could induce cell death in N. benthamiana leaves
To gain more insight into the function of TuRLK1, we transiently overexpressed TuRLK1 in N. benthamiana leaves. As shown in Fig. 3a, overexpression of the full-length TuRLK1 in N. benthamiana leaves induced hypersensitive cell death response (HR) in the injection region; however, the cell death phenotype was not as strong as that of the Pm60 protein, which is an NLR protein in PI428309, a control used [24]. By contrast, the NB-LRR domain of Pm60, which lacks the coiled-coil domain, did not induce cell death consistent with the previous reports (Fig. 3a). Additionally, HA-TuRLK1 protein was correctly expressed in N. benthamiana tested by immunoblot analysis (Fig. 3b).

Silencing of TuRLK1 in PI428309 compromised the resistance of YrU1 against Pst CY33
To study whether TuRLK1 is involved in the YrU1-conferred stripe rust resistance, we knockdown TuRLK1 via BSMV-induced gene silencing (VIGS) system in PI428309, the T. urartu accession that contains the functional YrU1. At 14 days post inoculation, the uredia were denser on the leaves with knockdown of TuRLK1 contrasting to those on the control leaves; nearly no necrotic spot caused by HR was observed, displaying more susceptible symptoms (Fig. 4a, b). Thus, silencing of TuRLK1 compromised the resistance of YrU1 to stripe rust Pst CYR33, and TuRLK1 is indispensable for stripe rust resistance conferred by YrU1.
To further characterize the stripe resistance on the leaves with reduced TuRLK1 expression, the infected leaves of PI428309 were collected at 2 days post inoculation (2 dpi), 3 dpi and 5 dpi, and wheat germ agglutinin (WGA) staining was used for visualizing the mycelium of Pst, which was observed by confocal microscope. We counted the number of hyphal branches (HB), haustorial mother cells (HMC) and haustoria (H) at 2 dpi, and the length of infection hyphae and the infection unit area were measured at 3 or 5 dpi, respectively. As shown in Fig. 4d, significant differences were existing in the number of HB, HMC except for H, between the TuRLK1 knockdown plants and the control samples (Fig. 4c). Similarly, in TuRLK1 silenced leaves, the length of infection hyphae and the infection unit area were significantly larger than those in the control leaves (Fig. 4e, f ). Taken all together, these results showed that TuRLK1 contributes to YrU1mediated stripe rust resistance.

The wheat powdery mildew resistance was potentiated after transient overexpression of TuRLK1
To investigate whether TuRLK1 is also involved in basal defense or disease resistance to other pathogens besides YrU1-mediated stripe rust resistance, we assessed the role of TuRLK1 on the wheat powdery mildew resistance using single-cell transient gene expression system mediated by particle bombardment. In this assay, we transiently expressed TuRLK1 in the susceptible bread wheat cultivar Fielder, and then inoculated the leaves with powdery mildew pathogen Blumeria graminis f. sp. tritici (Bgt) E09. As shown in Fig. 5a, the haustorium index in the leaves of Fielder was significantly decreased when transiently expressing TuRLK1 comparing to expression of PGY, a control used, which indicating that TuRLK1 plays an important role in resistance against Bgt E09. In addition, we examined the transcript levels of TuRLK1 in PI428309 by qRT-PCR at 0 hpi, 12 hpi, 24 hpi, 36 hpi, 48 hpi and 60 hpi, after Bgt E09 infection. As shown in Fig. 5b, the expression of TuRLK1 increased significantly after infection, which suggesting that TuRLK1 may play a role in the resistance to wheat powdery mildew too.

TuRLK1 enhanced the resistance to powdery mildew in Arabidopsis thaliana
To further investigate the function of TuRLK1 in disease resistance, we overexpressed TuRLK1 in Arabidopsis thaliana, and then assessed the powdery mildew resistance of the transgenic plants. In this experiment, TuRLK1 was fused with a HA tag mediated by 35S promoter, then, transformed into Arabidopsis thaliana accession Col-0. We inoculated two independent transgenic lines overexpressing TuRLK1 (TuRLK1-7 # , TuRLK1-10 # ) with fungus G. cichoracearum, which is the powdery mildew pathogen of Arabidopsis thaliana. The leaves of the two representing overexpressed lines showed much weaker growth of powdery mildew fungi in contrast to the control samples, and displayed some necrotic spots caused by cell death at 7 dpi (Fig. 6a, b). To quantify the fungal growth, we counted the number of conidiophores at 5 dpi. As shown in Fig. 6c, d, the conidiophores per colony in overexpressed plants were significantly less than that in control plants. Taken together, these results indicate that overexpression of TuRLK1 in Arabidopsis thaliana could enhance the resistance against G. cichoracearum and cause mild cell death in the leaves of transgenic plants.

Discussion
Many LRR-RLKs act as PRRs or PRRs co-receptors and play key roles in plant immunity [25]. In this study, we showed that TuRKL1 is involved in stripe rust resistance mediated by YrU1. TuRLK1 belongs to the LRR-RLK subfamily and contains only six LRRs in PI428309. Therefore, it is unlikely that TuRLK1 functions as a PRR, and it may rather function as a PRRs co-receptor or as a signaling protein in the immune complex that assists transmit the immune signal in PTI. Intriguingly, compared with the TuRLK1 in the T. urartu susceptible accession G1812, TuRLK1 in PI428309 has two additional LRR motifs in its ectodomain (Fig. 1c). It would be very interesting to investigate whether those TuRLK1 variants have distinct immune function. Previously, we showed that the transgenic plants of YrU1 in Bobwhite background are able to confer stripe rust resistance [15]. It is likely that Bobwhite has a homologous gene of TuRLK1 with a similar function. Consistent with this notion, a highly similar protein of TuRKL1 was identified in Chinese Spring (Additional file 1). It is interesting that TuRLK1, a PTI component, is required for ETI activated by NLR YrU1 after Pst CY33 infection. It is consistent with recent finding that ETI co-opts part of the PTI machinery as an indispensable component [8]. Nevertheless, how TuRLK1 functions in YrU1-mediated ETI is unclear. Besides TuRLK1, what other key immune components are required for the ETI triggered by YrU1 is also unknown. The recent studies in Arabidopsis indicated that a robust level of BIK1 and RBOHD, which mediates ETI ROS generation, full immunity-associated gene expression and disease resistance during ETI, is essential for functioning synergistically between the two primary classes of plant immune receptors, PRRs and NLRs [7,8]. Although, this conclusion was based on research regarding to bacterial disease and in Arabidopsis. However, it would be interesting to examine whether BIK1 and RBOHD are also the key immune components that contribute to the stripe rust resistance of YrU1 in wheat.
In addition to playing an important role in YrU1-mediated stripe rust resistance, TaRLK1 appears to confer broad resistance to other pathogens, as transient overexpression of TuRLK1 decreased the haustorium index and enhanced the resistance to powdery mildew in wheat. Furthermore, stable overexpression of TuRLK1 could also enhance the resistance against powdery mildew in Arabidopsis. One possibility is that TuRLK1 may act as a coreceptor or signaling protein that potentiates PTI. In this scenario, overexpression of TuRLK1 in wheat and Arabidopsis thaliana may enhance the function of associated PRR and result in production of reactive oxygen species (ROS), activation of mitogen-activated protein kinases (MAPKs) and induction of defense genes.
Although previously we showed that a NAC transcription factor TuNAC69 contributes to YrU1-mediated resistance in T. urartu [23], the components required for YrU1-mediated resistance are largely unknown. In this study, we identified an RLK, TuRLK1 that functions in plant immunity. TuRLK1 localizes in the plasma membrane and its expression is induced in Triticum urartu PI428309 after inoculation with Pst. We demonstrated that TuRLK1 is indispensable for the stripe rust resistance mediated by YrU1, an NLR with atypical domains, and TaRLK1 also enhances resistance to powdery mildew after overexpression in wheat and Arabidopsis thaliana. The results shed light on the understanding of the mechanism of immune response mediated by YrU1. Moreover, this study provides new evidence that PTI components are required for NLRmediated plant immunity, to the fungal disease resistance in wheat, besides Arabidopsis model plants. However,  some important questions are remained to be addressed,  including how TuRLK1-associated PRR immune complex  recognizes the pathogens and transduces the immune  signal; what are the PRRs interacting with TuRLK1, and  whether other key PTI components are required for YrU1-mediated stripe rust resistance.

Conclusion
Our study found that the expression of a leucine-rich repeat receptor-like kinase TuRLK1 was up-regulated after Pst CYR33 infection. TuRLK1 was plasma-membrane localized and could induce hypersensitive cell death response in N. benthamiana leaves. Transiently silencing TuRLK1 in diploid wheat Triticum urartu PI428309 that contains YrU1 by VIGS could compromise the resistance to stripe rust. Furthermore, overexpression of TuRLK1 in common wheat and Arabidopsis enhanced the resistance against powdery mildew. In summary, our work indicates that TuRLK1 is required for immune response to stripe rust mediated by NLR protein YrU1, and may also play an important role in disease resistance to other pathogens. This study provides new insight into the role of YrU1 and TuRLK1 in disease resistance, and may reveal potential connections between ETI and PTI in fungal disease.

Plant materials and growth conditions
The Triticum urartu accession PI428309, containing the stripe rust resistance gene YrU1, originated from El Beqaa, Lebanon, whereas the susceptible accession G1812 originated from Mardin, Turkey [24]. The winter bread wheat cultivar Kn199, which is highly susceptible to powdery mildew, was used to maintain the strain Bgt E09 for executing inoculation experiments. The winter bread wheat cultivar Mingxian169 was used to maintain the Pst CYR33 for the following assays. The foregoing materials were vernalized in preparation for sowing. The spring bread wheat cultivar Fielder is highly susceptible to Bgt E09, which is used in single-cell transient gene expression assay and maintaining Bgt E09. For phenotyping, these Triticum spp. plants were grown in the glasshouse at 20-22 °C under a 12-h: 12-h, light: dark photoperiod with about 60% relative humidity. T. urartu accession PI428309 and bread wheat cultivar Mingxian169, being inoculated with Pst CYR33, was cultured in the greenhouse at 16 °C under a 14-h: 10-h, light: dark photoperiod with about 80% relative humidity. Bread wheat cultivar Fielder inoculated with Bgt E09 was grown in an incubator at 22 °C under a 12-h-light:12h-dark photoperiod with about 70% relative humidity [23]. Arabidopsis thaliana and Nicotiana benthamiana plants were cultured in a growth room at 20-22 °C with a 9-h-light/15-h-dark cycle for phenotyping and a 16-h-light/8-h-dark cycle for seed setting, under light intensity of 7,000-8,000 lx [26,27]. Powdery mildew strain Golovinomyces cichoracearum UCSC1 was maintained with Arabidopsis mutant pad4, which was grown in an incubator after inoculation under the same conditions as infected Fielder [28].

Pathogen infection
Inoculation of Pst CYR33 was performed as described previously [15,29,30]. During the inoculation, talcum powder was mixed with urediospores as an indicator on the leaves. After inoculation, plants were brought to an incubator at 10 °C with 100% relative humidity in the dark for 24 h, immediately. Then, the plants were moved to a greenhouse under the conditions mentioned above. The stripe rust infection type (IT) was evaluated at 14 dpi, basing on a 0-4 scale: 0, immune, no visible uredia and necrosis on leaves; 0;, nearly immune, no uredia with hypersensitive flecks on leaves; 1, very resistant, few small uredia with distinct necrosis on leaves; 2, moderately resistant, few small-to medium sized uredia with dead or chlorosis on leaves; 3, moderately susceptible, a lot of medium-sized uredia, no necrosis, but with chlorosis on leaves; 4, highest susceptible, a large number of large-sized uredia without necrosis on leaves [15]. Plants with IT = 3-4 were susceptible. Inoculations of Bgt E09 and Golovinomyces cichoracearum UCSC1 were also performed according to previous publications [24,27,31].

RNA isolation, RNA-sequencing and assembling the reads
At the two-leaf stage, the first leaves of PI428309 were detached to extract RNA using the RNeasy Plant Mini Kit (cat. no./ID:74,903; Qiagen). An Agilent 2100 was employed to check the quality of RNA (Agilent Technologies Inc., Santa Clara, CA, USA). RNA-Seq was conducted as described previously [32][33][34], conducting by the Beijing Genomics Institute (BGI) using the BGISEQ-500 platform. All the reads were cleaned and assembled using the CLC GENOMIC WORKBENCH software (https:// www. qiage nbioi nform atics. com/ produ cts/ clc-genom ics-workb ench/).

Quantitative reverse transcription PCR (qRT-PCR)
Total RNA extraction and qRT-PCR were performed as previous publication [35]. M-MLV Reverse Transcriptase was used to reverse-transcribe total RNA (Promega, Madison, WI, USA). Transcript levels were examined by qRT-PCR on a BIO-RAD CFX Connect Real-Time System (Bio-Rad Laboratories, Hercules, CA, USA). ACTIN was used as the internal control and Student's t-test was performed to evaluate quantitative variation.

Staining and microscopy
In order to visualize cell death in N. benthamiana leaves, the infiltrated leaves were stained with trypan blue solution, boiled for 10 min, and decolorized overnight. Then, the cell death in N. benthamiana leaves after staining was directly photographed, using a digital single lens reflex camera [36].

Virus-induced TuRLK1 silencing
For silencing of TuRLK1, a 216-bp fragment of the gene was inserted in forward orientation into the Barley stripe mosaic virus RNAγ to form the recombinant vector BSMV: TuRLK1. The second fully expanded leaves of the PI428309 seedlings were infected with the recombinant vector, using BSMV: GFP (GFP, green fluorescent protein) as a control. The details of the assay were described in the previous publication [41]. Then, the fourth leaves were inoculated with Pst CY33 and evaluated the resistance at 14 dpi. The expression levels of TuRLK1 were determined by qRT-PCR [24].

Single-cell transient gene expression assay
The single-cell transient expression assay, using biolistic particle delivery of plasmid DNA into plant epidermal cells, was performed as previous publication [43]. The reporter plasmid containing the β-glucuronidase (GUS) gene and the plasmid pUBI: TuRLK1 were mixed before coating of the particles under the molar ratio of 1: 1, and the total DNA must be not more than 2.5 μg. Plasmid pUBI: PGY was used as control. The haustorium index was examined using microscopy by the mean of three independent experiments, observing at least 40 interactions in each repeat [24]. Single-cell transient gene expression method could also be used in subcellular localization assay.

Agrobacterium-mediated transformation of Arabidopsis thaliana
The 1980-bp coding domain sequence of TuRLK1 was cloned into binary vector pYBA 1143. The construct was introduced into the Agrobacterium tumefaciens strain GV3101. GV3101 with recombinant plasmid was suspended in a 5% (w/v) sucrose solution buffer containing 0.02% (v/v) Silwet L-77 and introduced the construct into Arabidopsis thaliana accession Columbia-0 (Col-0) through the floral dip method. The plants were cultured in a growth room under the conditions for seed setting. Positive individuals were screened on ½ Murashige and Skoog medium (½ MS) containing 0.005% (w/v) kanamycin [44,45]. The stable transformants were identified in the T 3 generation, which were used for further analysis.

Primers
Primers used in this study are list in Additional file 2.

Additional file 2. List of primers used in this study.
Additional file 3. Original image for Figure 3b.