Receptor-Like Kinase (RLK) as a candidate gene conferring resistance to Hemileia

: The biotrophic fungus Hemileia vastatrix causes coffee leaf rust (CLR), one of the most devastating diseases in Coffea arabica . Coffee, like other plants, has developed effective mechanisms to recognize and respond to infections caused by pathogens. Plant resistance gene analogs (RGAs) have been identified in certain plants as candidates for resistance ( R ) genes or membrane receptors that activate the R genes. The RGAs identified in different plants possess conserved domains that play specific roles in the fight against pathogens. Despite the importance of RGAs, in coffee plants these genes and other molecular mechanisms of disease resistance are still unknown. This study aimed to sequence and characterize candidate genes from coffee plants with the potential for involvement in resistance to H. vastatrix . Sequencing was performed based on a library of bacterial artificial chromosomes (BAC) of the coffee clone ‘Híbrido de Timor’ (HdT) CIFC 832/2 and screened using a functional marker. Two RGAs, HdT_ LRR_RLK1 and HdT_LRR_RLK2, containing the motif of leucine-rich repeat-like kinase (LRR-RLK) were identified. Based on the presence or absence of the HdT_LRR_RLK2 RGA in a number of differential coffee clones containing different combinations of the rust resistance gene, these RGAs did not correspond to any resistance gene already characterized (S H 1-9). These genes were also analyzed using qPCR and demonstrated a major expression peak at 24 h after inoculation in both the compatible and incompatible interactions between coffee and H. vastatrix . These results are valuable information for breeding programs aimed at developing CLR-resistant cultivars, in addition to enabling a better understanding of the interactions between coffee and H. vastatrix .


Introduction
For many centuries, rust diseases have been a major threat to many crops, causing severe damage to farming activities. In coffee (Coffea arabica), leaf rust, caused by the biotrophic fungus Hemileia vastatrix, is the most devastating disease worldwide (Zambolim, 2016). Resistant cultivars have been developed and successfully sustained in different coffee-producing regions of the world (Pestana et al., 2015;Alkimim et al., 2017;Valencia et al., 2017). However, owing to the high adaptive potential of the pathogen, the emergence of new physiological races and a corresponding 'breakdown' of resistance has been observed in many coffee cultivars (Várzea and Marques, 2005;Capucho et al., 2012;Cressey, 2013).
The pathogen, in general, employs several strategies to infect its host. One strategy is the secretion of effector proteins capable of suppressing plant defense responses and allowing the colonization of host tissues. In response to the infection, plants have evolved an innate immune system consisting of two lines of defense which limit the proliferation of pathogens in their tissues. Pattern-triggered immunity (PTI) is triggered by pathogen-associated molecular patterns (PAMP) and the second line of defense, effector-triggered immunity (ETI) fit the gene-for-gene theory proposed by Flor (1971), and (Dodds and Rathjen, 2010). This theory explains the interaction between coffee and H. vastatrix, whereby the resistance of coffee plants is conditioned by at least nine dominant genes with main effects (S H 1-S H 9). The S H 1, S H 2, S H 4, and S H 5 genes are found in C. arabica, whereas S H 6, S H 7, S H 8, and S H 9 are present in C. canephora and S H 3 is found in C. liberica (Noronha-Wagner and Bettencourt, 1967;Bettencourt and Noronha-Wagner, 1971;Bettencourt et al., 1980;Bettencourt and Rodrigues, 1988).
In our study, a novel and important candidate gene corresponding to a leucine-rich repeat (LRR) receptor-like serine/threonine-protein kinase was identified, and cloning was performed by screening a library of bacterial artificial chromosome (BAC) clones corresponding to 'Híbrido de Timor' (HdT) CIFC 832/2 (Cação et al., 2013). In addition, we developed functional markers linked to this receptor-like kinase (RLK) gene that can be used in marker-assisted selection in coffee breeding programs, allowing for early selection of individual and pyramidal genes for more durable resistance.

Screening of BACs
A library of 56,832 BAC clones derived from one of the main resistance sources, 'HdT' CIFC 832/2 (Cação et al., 2013), was used for screening the molecular marker HT24F133. This marker corresponds to the LRR receptor-like serine/threonine-protein kinase NIK1/ protein NSP interacting kinase 1-like, which was found to be upregulated in resistant coffee lines (Barka et al., 2017).
The BAC library was replicated in 384-well titration plates using a plate replicator sterilized in a laminar airflow hood. Old cultures were copied onto a new 384-well titration plate with 70 μL of fresh LB medium (containing 12.5 μg mL -1 chloramphenicol) in each well. Multiplication of the cultures was done by incubating them at 37 °C for 18 h on a shaker at 180 rpm. The plasmid DNA of the selected BAC library was screened using PCR with the molecular marker HT24F133. Clones were identified by grouping and subsequent group decomposition of the 384 clones until a single clone was identified as proposed by Cação et al. (2013). The plasmid DNA of the selected BAC clone 104-O-23 was extracted using a Wizard ® Plus SV Miniprep DNA purification kit (Promega, Madison, USA), following the manufacturer's recommendations.

New generation sequencing of the BAC clones and contig assembly
The DNA fragment isolated from the BAC library was sequenced in an Illumina HiSeq2000/2500 100PE platform (paired-end run with 101 bases per read), and analyzed using multiplexed shotgun sequencing. DNA samples were prepared for sequencing using Illumina's Nextera® XT DNA. Sequences were processed and contig-assembled using the SPAdes algorithm (version 2.5.1) (Bankevich et al., 2012).
The quality of each base call was determined by estimating a quality score similar to the phred score based on the image output without considering the reference sequence. More precisely, Bustard (version 1.8.28), estimates the P probability of a base call of being wrong and reports the corresponding quality score Q = -10 log 10 (P/(1−P)). A quality assessment of scaffolding assembly was performed using a QUAST Genome Assembly Assessment Tool (version 5.0.2) (Gurevich et al., 2013).

Prediction and annotation of genes
Gene prediction was formulated using the generalized hidden Markov model in AUGUSTUS (version 3.22), which provides a general feature format (GFF3) file containing information such as the position of genes, exons, introns, and transcripts for each gene (Stanke et al., 2004). Contigs longer than 500 bp were selected for gene prediction in AUGUSTUS (Stanke et al., 2004). Predicted open reading frames (ORFs) were annotated based on the detection of conserved domains in PFAM (version 32.0) protein families (Finn et al., 2014). The transmembrane domains of proteins were analyzed using the TMHMM Server v.2.0 (http://www.cbs.dtu.dk/ services/TMHMM/). To check for coding sequences, the predicted ORFs were subjected to a BLASTn (Basic Local Alignment Search Tool) (Johnson et al., 2008) search in the C. canephora (http://coffee-genome.org/blast) and C. arabica (https://blast.ncbi.nlm.nih.gov/blast) databases.
'Caturra Vermelho' CIFC 19/1 was used as the susceptible coffee line and 'HdT' CIFC 832/1 as the resistant line. The experiment was conducted in a growth chamber using a completely randomized design, with three biological replicates. One-year-old greenhousegrown young plants of 'Caturra' and 'HdT' were challenged with the pathogen race XXXIII of H. vastatrix as proposed by Capucho et al. (2009). The samples were collected at 0, 12, 24, and 96 h after inoculation (h.a.i).
For RNA extraction, the inoculated leaves were collected and macerated in liquid nitrogen. Total RNA was extracted using 100 mg of the macerated tissue with RNeasy Plant Mini Kit (Qiagen), following the manufacturer's recommendations. The RNA was quantified using Qubit RNA BR and a NanoDrop spectrophotometer. RNA integrity was evaluated using agarose gel electrophoresis (1.5 %), followed by staining with ethidium bromide. The samples were stored in an ultra-freezer at -80 °C until use.
The cDNA was synthesized with 3 μg of total RNA, which was pretreated with 1 μL of DNAse for 15 min (50 U μL -1 , amplification grade DNAse I to remove possible genomic DNA contamination. The first cDNA strand was synthesized using the ImProm-II™ Reverse Transcription system protocol RT-PCR kit, according to the manufacturer's guidelines and was subsequently stored at -20 °C until use. Real-time quantitative PCR was performed in a 7500 Real-Time PCR System. Reactions were induced in a final volume of 10 μL with 50 ng μL -1 of cDNA and 2 μM of forward and reverse primers in 1 × GoTaq qPCR Master Mix. The reaction conditions were: 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The melting curve stage was set to default conditions. The expression level of the genes was quantified by relating the threshold cycle value to a standard Gene resistance to Hemileia vastatrix Sci. Agric. v.78, n.6, e20200023, 2021 curve generated by three biological replicates and three technical replicates. For data normalization, we used two selected constitutive genes (UBQ10 and GAPDH) that had stable expression levels.
Statistical analyses were performed using Prism 6 (GraphPad Software, La Jolla, USA). All data are presented as the mean ± SEM, and, unless otherwise stated, p values (obtained using Student's t-test) < 0.05 were considered significant.

PCR amplification in coffee clones
For characterization of the identified gene HdT_ LRR_RLK2, a set of coffee clones bearing different resistance gene combinations was analyzed. These differential coffee clones proposed by the Coffee Rust Research Center (Centro de Investigação das Ferrugens do Cafeeiro, Portugal) have been used to characterize the physiological races of H. vastatrix (Várzea and Marques, 2005). We also included CIFC 832/2, the genotype that originated from the BAC library, as a positive control. 'Caturra' CIFC 19/1 and 'Catuaí Amarelo' IAC 64 (accession UFV 2148-57) were used as negative controls, as they are susceptible to all known Brazilian races of H. vastatrix. All clones were vegetatively propagated in the greenhouse in Viçosa, MG, Brazil (20°45'28.7" S, 42°52'11.7" W, altitude of 648 m above sea level).
Genomic DNA was extracted from the young second pair of leaves, following the protocol described by Diniz et al. (2012). DNA integrity was checked by subjecting the samples to electrophoresis in a 1 % gel, followed by staining with ethidium bromide (0.5 μg mL -1 ); DNA was quantified using a NanoDrop spectrophotometer and stored at -20 °C until use.
PCR was optimized such that the reaction mixture contained 50 ng of genomic DNA, 0.1 μM of each primer P2 to candidate gene HdT_LRR_RLK2, 0.15 mM of each dNTP (Promega), 1 mM MgCl 2 , 1 U of Taq DNA polymerase, and 1 × PCR reaction buffer, to yield a final volume of 20 μL. DNA was amplified in a thermocycler, programmed with initial denaturation at 94 °C for 5 min, followed by 34 amplification cycles of denaturation at 94 °C for 30 s, annealing at 66 °C for 30 s, an extension at 72 °C for 1 min and a final extension at 72 °C for 10 min. The amplification products were visualized after electrophoresis on a 6 % polyacrylamide denaturing gel, followed by the staining of the gel with silver nitrate.

BAC clone identification, sequence assembly, and gene prediction
The clone 104-O-23 (with a ~120 kb insert) was selected from the BAC library owing to the presence of the HT24F133 marker. This marker was developed to amplify a candidate gene identified by Barka et al. (2017), which corresponds to "LRR receptor-like serine/ threonine-protein kinase NIK1/protein NSP interacting kinase 1-like" and has the potential to be involved in the resistance of coffee to H. vastatrix. The BAC clone 104-O-23 was sequenced, and the assembled genomic region had an N50 value of 78.320 and L50 value of 20. The reads were assembled as 145 contigs, with 44.1 % GC content and 55.9 % AT content. In the 145 contigs, 1484 ORFs were found. The comparison with the C. canephora and C. arabica genomes allowed for the annotation of 37 ORFs in 10 contigs (Table 1).
Based on the annotation, contig 37 (16.570 bp), contig 51 (8.285 bp), and contig 65 (17.594 bp) revealed candidate genes related to host defense to pathogens. Aiming to identify the candidate gene involved with the resistance of coffee to rust as proposed by Barka et al. (2017), we selected contig 37 owing to the presence of genes coding the LRR motif.
Contig 37 had four genes ( Table 2). The gene g339.t1 denoted in this study as HdT_LRR_RLK1 is homologous to sequences in C. canephora and C. arabica (coverage > 50 %) on chromosome 1. Genes g340.t1 and g341.t1 have no homologs to any other genome; however, g342.t1 denoted as HdT_LRR_RLK2 is homologous to sequences in C. arabica on chromosome 1e and 1c (coverage > 50 %) ( Figure 1A, Table 1). The structures of genes HdT_LRR_RLK1 and HdT_LRR_RLK2 are represented in Figure 1B and C, respectively, showing promoter regions, presence of exons and introns, and anti-sense (3'-5') orientation.
The protein domains in the genes were identified using the PFAM database. HdT_LRR_RLK1 had four LRR domains, one leucine-rich repeat N terminal (LRRNT) domain and protein tyrosine kinases-Pkinase (TYR) domain ( Figure 1D). HdT_LRR_RLK2 had two LRR domains, and one cytochrome P450 and protein kinase-Pkinase domain each ( Figure 1E). These two genes have typical RLK domains, and in general, contain an extracellular domain, a single transmembrane domain, and a cytoplasmic kinase domain. LRR kinases represent a large and functionally diverse family of transmembrane proteins critical for signal recognition and transduction at the plant cell plasma membrane . The HdT_LRR_RLK1 and HdT_ LRR_RLK2 identified in the BAC clone had protein transmembrane helices, evident from the TMHMM analysis ( Figure 1D and E).

Gene expression analysis
The genes HdT_LRR_RLK1 and HdT_LRR_RLK2 had differential expression in both the compatible and incompatible coffee-H. vastatrix interactions (Figure 2). The expression peak of the gene HdT_LRR_RLK1 was observed at 24 h.a.i in the compatible interaction. The expression of HdT_LRR_RLK2 was significantly (p < 0.05) higher at 24 h.a.i in the incompatible interaction, as the appressorium and penetration hypha were differentiated. Based on these results, the HdT_LRR_ RLK2 gene was considered to be a candidate resistance gene.

Gene HdT_LRR_RLK2 in differential coffee clones
The presence of the gene HdT_LRR_RLK2 in the differential coffee clones was analyzed using the P2 primers. The marker was detected in four differential coffee clones, CIFC 832/2 (S H 6, S H 7, S H 8, S H 9, S H ?), CIFC 832/1 (S H 6, S H 7, S H 8, S H 9, S H ?), CIFC 1343/269 (S H 6), and H419/20 (S H 5, S H 6, S H 9) (Figure 3, Table 3). The S H 6 gene was present in these four clones, suggesting that S H 6 could be our candidate resistance gene. However, S H 6 had also been identified in the clones H420/10 and CIFC Gene resistance to Hemileia vastatrix Sci. Agric. v.78, n.6, e20200023, 2021 4106 although these clones do not have the HdT_LRR_ RLK2 marker. These results indicate that the candidate cloned gene does not correspond to any of the S H genes previously identified in the set of differential coffee clones (S H 1 to S H 9).  Error bars = three independent biological replicates. * Shows significant difference in the expression levels at the same h.a.i between the interactions, * shows significantly upregulated or downregulated expression levels relative to the uninoculated samples (0 h.a.i).

Discussion
Several genetic studies have identified coffee genes associated with rust resistance. At least nine genes (S H 1 to S H 9) have been inferred according to the Flor (1971)  The gene HdT_LRR_RLK2 has domains typical of plant RLKs. RLKs are the major components of pattern-recognition receptor (PRR) complexes that comprise a superfamily of transmembrane proteins, many of which function in pathogen detection as PRRs (Tang et al., 2015). Plants have evolved various defense mechanisms to combat diseases. The plant's innate immune system mainly consists mainly of two interconnected branches, termed pattern-triggered immunity and effector-triggered immunity (Jones and Dangl, 2006;Cui et al., 2015;Boutrot and Zipfel, 2017).
Leucine-rich repeat-receptor-like kinases (LRR-RLKs), which contain up to 30 LRRs in their extracellular domain, constitute the largest RLK family (Shiu and Bleecker, 2001). The LRR_RLK gene family contributes to basal immunity to adapted pathogens and to nonhost resistance to non-adapted pathogens through the induction of both local and systemic immune responses with broad-spectrum and potentially durable disease resistance (Boutrot and Zipfel, 2017;Hu et al., 2018;Ranf, 2018).
Based on the candidate resistant gene identified, a functional molecular marker was developed. After validation of this marker in a segregated coffee population for resistance to H. vastatrix, the functional marker can be efficiently used for marker-assisted selection of resistant seedlings in coffee breeding programs. gene-for-gene theory (Noronha-Wagner and Bettencourt, 1967;Bettencourt and Noronha-Wagner, 1971;Bettencourt et al., 1980;Bettencourt and Rodrigues, 1988). However, the genes that participate in the coffee resistance mechanism have not yet been studied at the molecular level; only S H 3 (Prakash et al., 2004;Mahé et al., 2007;Ribas et al., 2011) was characterized at the molecular level. In our study, a disease resistance gene analogue was cloned and characterized (gene HdT_LRR_ RLK2). The analysis of the presence/absence of this gene in a set of differential coffee clones (Table 3) suggested that the cloned candidate gene is not one of the nine S H genes reported previously. The cloning of genes in our study was based on the data reported by Barka et al. (2017), who identified an "LRR receptor-like serine/threonine-protein kinase NIK1/protein NSP interacting kinase 1-like" with potential association to coffee rust resistance. By screening a BAC library, a BAC containing this candidate gene was sequenced and two genes encoding the LRR protein motif were identified and characterized.
To analyze the association of the cloned genes in coffee rust resistance, an RT-qPCR study was conducted, to consider the compatible and incompatible interactions between coffee and H. vastatrix. The HdT_LRR_RLK1 gene was expressed at 24 h.a.i in the susceptible genotype, whereas the HdT_LRR_RLK2 gene was expressed at 24 h.a.i in the resistant genotype. At 24 h.a.i, H. vastatrix infection of coffee leaves involved the formation of the appressoria, after which the fungus penetrated the stomata, forming a penetration hypha that grew, entered the substomatal chamber (Florez et al., 2017;Talhinhas et al., 2017). Florez et al. (2017), and used the same pathosystem, 'Híbrido de Timor' CIFC 832/1 (resistant) and 'Caturra' CIFC 19/1 (susceptible), and confirmed that early expression patterns of upregulated genes in 'HdT' are directly related to prehaustorial resistance. Diniz et al. (2012) reported that rapid resistance response preventing the formation of haustoria might be the basis for the extended durability of the resistance of 'HdT' CIFC 832 to H. vastatrix races. In our study, we showed that HdT_ LRR_RLK2 expression peaked in the resistant coffee lines at 24 h.a.i, suggesting that it can be a candidate resistance gene capable of inducing immune signaling of and defense in coffee plants against H. vastatrix.