81 J. Hortl. Sci. Vol. 15(1) : 81-92, 2020 Original Research Paper Identification of NBS-LRR Resistance Gene Analogues (RGA) from Rose (IIHRR13-4) Resistant to Powdery Mildew (Podosphaera pannosa (Wallr.:Fr.) de Bary) Neethu K. Chandran1, 3, Sriram S.1*, Tejaswini Prakash2 1Division of Crop Protection, 2Division of Floriculture and Medicinal crops, ICAR-Indian Institute of Horticultural Research, Bengaluru - 560 089, India 3Jain University, Bengaluru - 560 089, Karnataka, India Email : Subbaraman.Sriram@icar.gov.in ABSTRACT Resistance is the best strategy to manage powdery mildew (Podosphaera pannosa (Wallr.:Fr.) de Bary) of rose. Identification of resistant genes (R genes) from plant species will help in breeding programs. Nucleotide Binding Site - Leucine Rich Repeats (NBS- LRR) is a major class of R gene family in plants. This study reports the identification and molecular characterization of resistance gene analogues from roses maintained at ICAR- Indian Institute of Horticultural Research (IIHR). The powdery mildew resistant line IIHRR13-4 was compared with the susceptible commercial cultivar, konfetti. PCR based approaches with degenerative primers based on different conserved motifs of NBS-LRR were employed to isolate resistance gene analogues (RGAs) from rose. Eleven RGAs (IIHRR13-4R1, IIHRR13-4R2, IIHRR13-4R3, IIHRR13-4R4, IIHRR13-4R5, IIHRR13- 4R6, IIHRR13-4R7, IIHRR13-4R8 IIHRR13-4R9 and IIHRR13-4R10) were identified from powdery mildew resistant germplasm line, IIHRR13-4, based on the sequence and similarity to RGAs from rosaceae family and other crops. The major similarity to rose RGAs reported are from Fragaria vesca, Rosa hybrid cultivar, Prunus and Rosa chinensis. RGAs isolated from IIHRR13-4 belonged to Toll Interleukin Receptor (TIR)-NBS-LRR and Non-TIR-NBS-LRR RGAs (Lecine Zipper (LZ) type). Different motifs of RGAs identified were P-loop, RNBS A, kinase 2, kinase 3a, RNBS-D and GLPL of NBS domain. This study reports the existence of resistance at genetic level in powdery mildew resistant genotype IIHRR13-4. These RGAs will be useful for mapping and characterization of R genes in IIHRR13-4 and breeding for improved powdery mildew resistance in roses. Key words: Nucleotide Binding Site-Leucine Rich Repeats (NBS-LRR), Podosphaera pannosa, Powdery mildew, Resistance Gene Analogues (RGA) and Rose. INTRODUCTION Two major events involved in defense mechanism are recognition of pathogen attack and induction of defense r esponses fr om pla nts a ga inst the pathogen.The defense response against particular pathogen is triggered by an interaction between R gene from the host and avirulence gene product of pathogen which restricts the pathogen invasion (Flor, 1971; Holt et al.,2000). R genes form a diverse group of related sequences that are widely distributed in plant genome. There are five classes of R genes based on their structural characteristics of predicted protein structure and majority of these R-genes belong to nucleotide-binding site (NBS) and leucine-rich repeats (LRRs) groups (Ellis and Jones, 1998; Hammond- Kosack and Jones, 1998; Hattendorf and Debener, 2007a; Hattendorf and Debener, 2007b). Putative NBS domains are concerned with signalling and they are characterized by several highly conserved motifs, viz. P-loop, Kinase-2 and Gly-Leu-Pro-Leu (GLPL) motifs. Structural domains of LRRs are involved in protein-protein interactions and pathogen recognition (Belkhadir et al., 2004; Ellis et al., 2003; Yung, 2000). This is an open access article d istributed under the terms of Creative Commons Attribution-NonCommer cial-ShareAl ike 4.0 International License, which permits unrestricted non-commercial use, d istribution, and reproduction in any med ium, provide d the original author and source are credited. 82 Chandran et al. J. Hortl. Sci. Vol. 15(1) : 81-92, 2020 The NBS-LRR domain is classified into two groups based on their N-terminus. First is the amino terminal toll interleukin receptor (TIR)-NBS-LRR and the second is without TIR region known as Non-TIR- NBS-LRR class. TIR-NBS-LRR is characterized by their similarity to toll receptor in Drosophila and interleukin 1 receptor of mammals and Non-TIR groups has a leucine zipper (LZ) or coiled coil(CC) motif instead of TIR. Non-TIR group is widely distributed in both monocots and dicots while TIR group is rare in cereals and grasses. So far several R genes have been cloned in different crops(Collier and Moffett, 2009; Dangl and Jones, 2001; Ellis et al., 2003; Hammond-Kosack and Jones, 1997; Jones, 2001; Liu et al., 2007). Resistance gene analogues are putative derivatives of R genes. The highly conserved domains provide unique identity to R genes in pla nt genome (Hammond- Kossack et al., 1996). The conserved motifs of NBS-LRR can be used to isolate resistance genes from plants by PCR based approach with oligonucleotide degenerate primers. RGAs have been isola ted fr om wide va r ieties of pla nt species (Hattendorf and Debener, 2007a). Characterization of RGA is an effective strategyto identify R genes and develop markers for disease resistance (Mayer et al., 1999; Hattendorf and Debener, 2007a; Biber et al., 2010; Backiyarani et al., 2013; Lei et al.,2014; Sekhwal et al., 2015). Rose is one of the economically important ornamental crops from Rosaceae family and it has the highest economic impact in the world. Rose flower industry comprises of local and international marketing of cut flowers, loose flowers, scent, oil and medicines. The worldwide estimated production of rose is 18 billion cut stems, 60-80 million potted-rose and 220 million for landscape purposes (Debener and Byrne, 2014). Apart from roses, other economically important members of Rosaceae family are apples (Malus), strawberries (Fragaria), stone fruits like peach, plum, apricots (Prunus) and pears (Pyrus). Most of the species of Rosaceae family are woody perennials. There is a wide range of pathogens viz., fungi, bacteria, virus, phytoplasma and nematodes that attacks rose plants causing its death and thereby reducing the marketability of roses. Powdery mildew is one of the most damaging diseases of Rosaceae family (Xu et al., 2007). Podosphaera pannosa (Wallr.:Fr.) de Bary is an obligate (biotrophic) pathogen (order Erysiphales, phylum Ascomycotina) that inhabits numer ous economically impor tant plants.Severe powdery mildew infection reduces greenhouse cut flower production (Leuset al., 2003; Xu et al., 2005, Debener and Byrne, 2014). Characterization of R genes from wild varieties will help in obtaining disease resistant cultivars of roses (Hattendorf et al., 2004; Hattendorf and Debener, 2007b). So fa r, only two R genes ha ve been characterized in rose viz. Rdr 1 for black spot resistance (Von Malek et al., 2000; Ayana et al., 2011) and RPP1 for powdery mildew resistance (Linde and Debener, 2003; Linde et al., 2004) from Institute for Ornamental Plant Breeding, Germany. Diseaser esistance loci have been identified and mapped in apple (Calengeet al., 2005; Perazzolli et al., 2014; Pessina et al., 2014), strawberries (Zamora et al., 2004), peach (Dirlewanger et al., 1996, 2004; Quarta et al., 2000; Dettori et al., 2001; Lalli et al., 2005), Arabidopsis thaliana (Aarts et al., 1998; Mayer et al.,2003) and soybeans (Yu et al.,1996). Study of R gene and its locus can help to reveal their exact function in pathogen recognition followed by defense and their evolution among particular plant species. This can be used to develop novel disease management strategies (McHale et al., 2006). In this context, the best desirable strategy for disease management is development of resistant varieties as it can be a cost effective alternative for chemical method of disease management. Powdery mildew resistance was observed in rose genotype (IIHRR13- 4) during field evaluation but mechanism of disease resistance was unknown. The objective of this study was to identify and characterize resistance gene analogues from IIHRR13-4. MATERIALS AND METHODS Rose genotypes used in this study were obtained from Division of Ornamental crops, ICAR- Indian Institute of Hor ticultur a l Resea r ch (IIHR). Eight r ose genotypes (Table 1) were used to identify resistant gene analogues based on earlier reports. Among those genotypes selected, IIHRR13-4 was found to be resistant, Rosa indica was immune and remaining were highly susceptible to powdery mildew. Genomic DNA was isolated from coppery red rose leaves by CTAB method (Doyle and Doyle, 1987). Six sets of degenerative primers (Table 2) were used 83 S.No. Rose genotypes used in the study Field assessment of Powdery mildew disease 1 R1 - IIHRR13-4 (PMR) Resistant 2 R2 - Rosa indica Resistant 3 R3 - 11-3 Susceptible 4 R4 - First Red Susceptible 5 R5 - Dean De Pointers Susceptible 6 R6 - Fantasy Susceptible 7 R7 - Konfetti Susceptible 8 R8 -13-24 Susceptible Table 1. Rose genotypes maintained at IIHR used in the study Table 2. List of primers used in this study for the amplification of RGAs S.No. Primer name Sequence (5’-3’) Motif 1 RS1 F GGIGGIATIGGIAAAACIAC GGMGKTT RS1 F RAARCAIGCDATRTGIARRAA FLHIACF 2 RS3 F GGIGTIGGIAAIACI GGVGKTT RS3 R RAARCAIGCDATRTGIARRAA FLHIACF 3 RS4F GGIGGIATIGGIAAAACIAC GGMGKTT RS4R RAARCAIGCSATRTCIARRAA FLDIACF 4 RS10F GGIGGIATIGGIAAAACIAC GGMGKTT RS10R YTCIGGRAAIARIGCRCARTA YCALFPE 5 RS11F GGIGGIYTIGGIAARACIAC GGLGKTT RS11R YTIIGGRAAIARIGCRCARTA YCALFPE 6 RS12F GGIGGIGTIGGIAAIACI GGVGKTT RS12F YTCIGGRAAIARIGCRCARTA YCALFPE for amplification of RGAs. RS1 RS2 and RS3 primer pairs were specific for TIR-NBS-LRR type and RS10, RS11 and RS12 were for LZ type (Hattendorf and Debener, 2007a). PCR assays were performed with genomic DNA in a total volume of 25 µl containing10µM of forward and reverse primers (Sigma Aldrich, India), 3 units of Taq polymerase and 2.5 mMTaq buffer (Genei, Bengaluru, India).PCR reaction was performed in Eppendorf thermal cycler with initial denaturation at 940C for 3 min, 35 cycles of 940C for 1 min, 420C for 1 min, 720C for I min followed by final extension step at 72 0C for 10 minutes (Hattendorf and Debener, 2007a). Agarose gel (1.5%) electrophoresis was carried out to view and purify the PCR products.The amplified products were further purified by Nucleospin gel and purification kit by Macherey-Nagel GmbH & Co. KG, Germany. The purified products were ligated into PTZ 57R/Tvector. Cloning was done with Thermo-Fisher Scientific InsTA clone PCR cloning kit (Thermo- Fisher Scientific Ba ltics UAB, Lithua nia ). Transformed colonies were selected for plasmid isolation and presence of insert was confirmed by plasmid PCR. Cloned plasmids were sequenced for further identification (Hattendorf and Debener, 2007a). RGA sequences wer e a na lysed using NCBI Vecscreen (https://www.ncbi.nlm. nih.gov/tools/ vecscreen/) and BioEdit (Hall, 1999). Sequence similarity search was done using NCBI Blast (https:/ J. Hortl. Sci. Vol. 15(1) : 81-92, 2020 Resistance gene analogues (RGA) in rose 84 /blast.ncbi.nlm.nih.gov) for RGA sequences. Amino a cid sequences wer e gener a ted using Expert Protein Analysis System (ExPASy) (https:// web. expa sy.org/tr anslate/) tra nsla ting tool a nd conserved motifs were identified by amino acid sequence a lignment. Phylogenetic tr ee wa s constructed with MEGA-6 (Tamuraet al., 2013) with bootstrap analysis with 1000 replications. Sequences of selected RGAs were deposited at NCBI-Gen Bank database. RESULTS AND DISCUSSION Genomic RGAs were identified from rose genotypes with various degenerate primer sets with an amplified product of 550-700 bp length (Fig.1).From the different primer sets used only RS1 and RS10 primer combinations amplified in rose plants irrespective of susceptibility or resistance. The PCR amplified products were cloned and 41 colonies were picked for confirmation and sequence analysis. Finally ten RGA clones were selected from IIHRR13-4 by RS1 pr imer combina tion a nd three RGAs identified respectively from IIHRR13-4, Konfetti and First red by RS10 combination. These RGAs were finalised based on the sequence length and similarity to RGAs from Rosaceae family and other plant RGAs. The RGAs sequences with internal stop codons were eliminated. Total eleven RGAs were confirmed from IIHRR13-4 after sequence analysis and similarity to other plant RGAs. Other two RGAs were confirmed Fig. 1. Agarose gel electrophoresis confirming the amplification of Rose RGAs fragments, 1-5, 1 -7 - Rose RGAs. RGA fragments amplified at 550-700bp. from powdery mildew susceptible First red and konfetti. All the amplified PCR pr oducts were purifiedand cloned in P TZ vector. The sequence homology of rose RGAs to other plant proteins and other known R genes was confir med by NCBI BLAST search. The list of proteins present in other plants belonging to rosaceae family to which close similarity was observed for the RGAs identified in the present study is given in Table 3. The R gene sequences retrieved from NCBI database used in the phylogenetic analysis are listed in the Table 4. RGAs identified in the present study showed similarity to both TIR class of NBS-LRR RGAs and Non-TIR (LZ) class of NBS-LRR. RGAs identified from susceptible varieties showed similarity to RGAs of Rosaceae family but some of them excluded after amino acid translation because of the presence of internal stop codons. Finally thirteen RGA sequences viz., IIHRR13-4R1, IIHRR13-4R2, IIHRR13-4R3, IIHRR13-4R4, IIHRR13-4R5, IIHRR13-4R6, IIHRR13-4R7, IIHRR13-4R 8, IIHRR13-4R9, IIHRR13-4R10, IIHRR13-4RS10 (IIHRR13-4), IIHRSFRR10 (First Red) and IIHRRRIRS10 (Rosa indica) were identified in this study. Multiple sequence a lignment identified highly conserved amino acid motifs present in the RGAs of IIHRR13-4. Multiple sequence alignment of IIHRR13- 4 RGAs was performed with other R genes of Rose, Arabidopsis, Solanum, Nicotiana, Malus, Prunus, Fragaria a nd a poptotic pr otea se a ctiva ting factor(APAF) gene (Fig. 2). Six highly conserved amino acids motifs of NBS domain identified were P- loop, RNBS (Resistance nucleotide binding Site)-A, Kinase-2, Kinase-3a, RNBS-D, and GLPL. NCBI CD-search (Conserved Domain software) was used to find and confirm the conserved domains of RGAs and presence of nucleotide binding domain (NBARC domain) a nd LRR3 super fa mily domain. The selected RGAs wer e further a nalysed for their phylogenetic relationships among Rosaceae family and other plant R genes. Chandran et al. J. Hortl. Sci. Vol. 15(1) : 81-92, 2020 85 Table 3. List of RGAs identified from the present study, their GenBank accession numbers and sequence similarity with RGAs of other Rosaceae family Rose NCBI Protein to which closer similarity Plant species Identity RGAs Accession observed (%) number IIHRR13- MG958641 TMV resistance protein N-like isoform X2 Fragaria vesca 77 4R1 sub sp. vesca Putative NBS-LRR resistance protein Rosa hybrid cultivar 80 IIHRR13- MG970527 Putative NBS-LRR resistance protein Rosa hybrid cultivar 98 4R2 Putative winged helix-turn-helix DNA- Rosa chinensis binding domain, leucine-rich repeat domain IIHRR13- MG970528 Putative transcription factor WRKY family Rosa chinensis 94 4R3 Putative TIR-NBS-LRR resistance protein Rosa hybrid cultivar 82 IIHRR13- MG970529 Putative transcription factor WRKY family Rosa chinensis 100 4R4 Putative TIR-NBS-LRR resistance protein Rosa hybrid cultivar 83 IIHRR MG970530 TMV resistance protein N-like Fragaria vesca subsp. 67 13-4R5 vesca TMV resistance protein N-like Prunus avium 62 IIHRR13- MG970531 Putative NBS-LRR resistance protein Rosa hybrid cultivar 87 4R6 Putative toll-like receptor, P-loop containing Rosa chinensis 88 nucleoside triphosphate hydrolase IIHRR13- MG970532 Putative TIR-NBS-LRR resistance protein Rosa hybrid cultivar 100 4R7 TMV resistance protein N-like Fragaria vescasu bsp. 73 vesca IIHRR13- MG970533 Putative TIR-NBS-LRR resistance protein Rosa hybrid cultivar 83 4R8 Putative transcription factor WRKY family Rosa chinensis 72 IIHRR13- MG970534 Putative TIR-NBS-LRR resistance protein Rosa hybrid cultivar 88 4R9 Putative transcription factor WRKY family Rosa chinensis 75 IIHRR13- MG970535 NBS-LRR resistance protein Rosa hybridc ultivar 88 4R10 Putative TIR-NBS-LRR resistance protein Rosa hybrid cultivar 84 IIHRR13- MK704433 Putative disease resistance protein Rosa chinensis 74 4RS10 RGA3 and RGA4 IIHRSFR MK704434 Putative disease resistance protein RGA1, Rosa chinensis 86, 85, 86 R10 (First RGA2, RGA3 and RGA4 and 90 Red) Isolate F11P2-4F NBS-LRR resistance Rosa hybrid cultivar 87 protein gene IIHRRRI MK689860 Putative disease resistance protein RGA2 Rosa chinensis 97, 88 RS10 RGA3 and RGA4 and 87 (Rosa NBS-LRR resistance protein gene Rosa hybrid cultivar 88 indica) J. Hortl. Sci. Vol. 15(1) : 81-92, 2020 Resistance gene analogues (RGA) in rose 86 Chandran et al. J. Hortl. Sci. Vol. 15(1) : 81-92, 2020 F ig . 2 . M ul tip le s eq ue nc e al ig nm en t s ho w in g co ns er ve d m ot if s in th e am in o ac id a lig nm en t o f II H R R 13 -4 R G A s w ith ot he r R g en es o f R os e, A ra bi do ps is , S ol an um , N ic ot ia na , M al us , P ru nu s, F ra ga ri a an d A PF g en e. H ig hl y co ns er ve d am in o ac id s of m ot ifs (P -l oo p, R N B S- A , K in as e- 2, K in as e- 3a , R N B S- D , a nd G LP L) a re s ha de d 87 J. Hortl. Sci. Vol. 15(1) : 81-92, 2020 Resistance gene analogues (RGA) in rose Phylogenetic tree was constructed using MEGA-6 software to identify genetic relationship and diversity among rose RGAs and other known plant R genes from Rosaceae and other species (Table 3). Different R genes from different crops available in GenBank were selected to analyse the phylogenic relationship among R genes. The phylogeny was constructed using Neighbour Joining method with1000 boot strap replications (Fig. 3). The two distinct groups of RGAs, T IR a nd LZ types wer e clea r ly sepa r a ted in phylogram. Apoptotic protease activating factor 1 (APAF1) related to human cell death was used as out-group to construct phylogentic tree because of its NBS domain with greater protein sequence similarity to NBS-LRR proteins of plants. Highest degree of similarity of IIHRR13-4 RGAs was observed with Malus domestica, Rosa chinensis, F. vesca and Rosa hybrid cultivar. Comparative sequence analysis specifies the clustering of rose RGAs with certain Rosaceae RGAs. IIHRR13-4R2, IIHRR13-4R3, IIHRR13-4R4, IIHRR13-4R8 andIIHRR13-4R9 clustered together with TIR-NBS-LRR Rosa hybrid cultiva r. IIHRR13-4R7 cluster ed with T MV r esista nce N like pr otein of Fragaria vesca. IIHRR13-4R5 clustered with TMV resistance N like protein of Prunus avium and TIR-NBS-LRR Rosa hybrid cultivar. IIHRR13-4R1 grouped with TMV of Nicotiana tabaccum. Other RGA IIHRRCONRS10 grouped with LZ-NBS-LRR of Rosa hybrid cultivar, NBARC of Arabidopsis thaliana , RGC1 of Solanum a nd RGA 4 of Rosa chinensis. RGA identified from Fir st Red IIHRSFRRS10 were grouped with Xa21 of Oryza sativa. Neighbour Joining phylogenetic tree confirms the similarity of TIR and LZ type of RGAs from IIHR rose genotypes to RGAs of Rosaceae family. Identification of RGAs can assist in breeding program for superior disease resistance because of the specific gene feature. RGA fragments are generated from different motifs of conserved domains of R genes that code for resistance against particular pathogen. RGA based markers that linked to R genes are more specific and facilitate selection of desirable disease resistant lines (Ellis et al., 2000; Biber et al., 2010, Hattendorf and Debener, 2007a). PCR based approach with degener a te pr imer is a n efficient method for identification and cloning of RGAs from plants (Hattendorf and Debener, 2007a; Vossen et al., 2013; Yu et al., 1996). PCR based approach was used in the study to identify potential RGAs linked to powdery mildew resistance in IIHR line of r ose (IIHRR13-4) for which molecular basis for mechanism of disease resistance had not been earlier identified. Based on previous obser va tions under field eva lua tion, genotype IIHRR13-4 that wasfound resistant was selected along with wild rose species R. indica which was Sl.No. R gene Host Accession No. 1 RGC1 Solanum tuberosum AF266747.1 2 NB-ARC domain disease resistance protein Arabidopsis thaliana NP187360.1 3 Virus resistance (N) gene Nicotiana glutinosa U15605.1 4 TIR-NBS-LRR type R protein 7 Malus baccata AAQ93075.1 5 TIR-NBS-LRR resistance protein Rosa hybrid AM075235.1 7 Rust resistance protein M gene Linum usitatissimum U73916.1 8 RPP5 Arabidopsis thaliana NM114316.3 9 L6 Linum usitatissimum U27081.1 10 RPW8.1, RPW8.2 Arabidopsis thaliana AF273059.1 11 Xa21 Oryza sativa  AY885788.1 12 LZ-NBS-LRR resistance protein Rosa hybrid AM075248.1 13 TMV resistance protein N-like Fragaria vesca subsp. vesca XM011459053.1 14 TMV resistance protein N-like Prunus avium XM021944693.1 15 Apoptotic protease activating factor 1 (APAF1) mRNA (out group) Homo sapiens  AF149794.1 Table 4. List of R genes retrieved from NCBI database used in the phylogenetic analysis that were compared with RGAs of rose 88 immune to powdery mildew. Molecular profiling of RGAs with NBS conserved motifs helps in diversity studies of R gene families and identification of molecular markers for disease resistance (R) genes (Vossen et al., 2013). Six degenerate primers of conserved NBS motifs used in the present study were selected from Hattendorf and Debener (2007) for isolation of genomic RGAs from rose. RS1, RS2 and RS3 primers helped in identifying TIR class of genomic RGAs and RS4, RS5 and RS6 aided amplification of non-T IR cla ss (LZ) of RGAs fr om r ose. T he difference in each primer set relies on single amino acid in the motif sequence. Primers of P-loop motif sequence (GG.GKTT) differed in the third amino acid of the motif (GG (M/V/L) GKTT). In the same way, primers designed on NBS-IX motif also differed in single amino acid change. Motif sequence of NBS- IX was FL.IACF and change was on the third amino acid (FL(H/D)IACF). These variation in primer sequences helps to identify complete set of RGAs present in the rose genome. Genomic RGAs isolated from rose belonged to TIR and non - TIR class (LZ) of NBS-LRR resistance genes. Non-TIR classes of RGAs are present in monocotyledons (wheat, rice, maize) and di-cotyledons but TIR class of RGAs mostly found in dicotyledonous plants. Hattendorf and Debener (2007a) reported that rose genome contained more TIR class of RGAs rather than non-TIR class with respect to number and diver sity of RGAs in r ose genome. T he clea r distinction between TIR and LZ class is based on the motif sequences of NBS domain (Xu et al., 2005). RGAs identified from all genotypes of rose (Table 1) ir respective of powdery mildew resistance a nd susceptibility. Pr evious investigations showed constitutive expression of RGAs but their transcription was induced by different factors in different crops Chandran et al. J. Hortl. Sci. Vol. 15(1) : 81-92, 2020 Fig. 3. The phylogenetic tree constructed with Neighbor-Joining method based on the amino acid sequences of rose RGAs, along with RGAs and R genes from Rosaceae and other plant species. The bootstrap values obtained from 1000 replications Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. 89 (Hammond-Kosack and Jones, 1997). Expression of RGAs specifically after pathogen attack explains their role particularly in defense response. Hattendorf and Debener (2007b) explained relative expression of RGAs in rose by checking the expression of RGAs in Diplocarpon rosae (black spot disease) inoculated and control rose leaves. Enhanced expressions of TIR- RGAs wer e obser ved in r ose a fter bla ck spot inoculation than untreated control, indicating direct function of TIR-RGAs in disease resistance against black spot. Genomic RGAs isolated from powdery mildew susceptible lines of rose (Table 1) were excluded because of presence of premature stop codons. RGA isola ted fr om Fir st Red (IIHRRSFRRS10) was without stop codons and that grouped with Xa21 of O. sativa in the phylogram. The genomic RGA of First Red probably may not express during powdery mildew infection process to prevent the disease and leads to the susceptibility to powdery mildew. NCBI blast results indicate that IIHRSFRR10 was showing similarity to Rosa hybrid cultivar NBS-LRR resistance protein pseudogene by 86.59%. Therefore, the RGA was identified from First Red ma y be pseudogene. T hese r esults ga ve information regarding the expression levels of RGAs in rose with respect to disease resistance. Some of the RGAs were identified as pseudogenes in many crops (potato, Arabidopsis, cotton, lotus and tomato). Non-functional pseudogene paralogs of R-genes (Xa21, Cf9, Pto and Dm3) were identified and have strong identity with other NBS protein but their sequences are short and presence of premature stop codons was observed. Pseudogenes are assumed to be involved in the R gene evolution process (Sekhwal et al., 2005; Songet al., 1997). The NCBI blast results showed that IIHRR13-4R4 were 100% similar to putative transcription factor WRKY family of R. chinensis. WRKY super family of plant transcription factors plays important role in plant defense. The plant immune receptors detect pa thogen effector pr oteins thr ough WRKY transcription factors and activate defense (Phukanet al., 2016). The NBS-LRR usually connects with other protein domains. Arabidopsis RRS1-RNB-LRR protein carries C-terminal WRKY DNA binding domain that enables formation of receptor complex with another NBS-LRR protein, RPS4 that helps in detection of bacterial effectors. This ligand-receptor binding initiates activation of defense mechanisms this indicates that the plants defense depends on intra- cellular immune receptors. The phylogenetic tree showed clear separation of two different classes of NBS-LRR RGAs (TIR and LZ). Resistance gene analogues identified in the present study were closely related to Rosa hybrid cultivar, R. chinensis and other species of Rosaceae family (Fragaria, Prunus and Malus). The conserved motifs identified from RGAs of IIHR13-4were similar to Rosa multiflora hybrid (Hattendorf and Debener, 2007a), chestnut rose (Xu et al., 2005), strawberry (Zamora et al., 2004) and other plant NBS-LRR R genes. These conserved motifs of NBS domain codesfor ATP or GTP binding proteins and hydrolysis activity (Phukan et al., 2016; Saraste et al., 1997; Xu et al., 2005). IIHRR13-4 RGAs were showing more homology with TIR-NBS-LRR disease resistance gene of Rosa hybrid and TMV- N like disea se resistance gene of F. vesca. The two major clades observed were TIR group and non-TIR group (LZ- NBS-LRR) of RGAs. TIR group of NBS-LRR genes of Rosa hybrid was clustered together with IIHRR13- 4 RGAs. Phylogenetic studies r evea led the relationship between RGAs identified from rose and other R genes/RGAs of Rosaceae and other family also. Multiple sequence alignment by ClustalW showed that different motifs of rose RGAs were P-loop, RNBS- A, kinase 2, kinase 3a, RNBS-C and GLPL of NBS domain. Rose TIR - RGAs carried an aspartic acid residue (D) at the end of kinase 2 region (NBS III). Usua lly LZ-RGAs (Leucine Zipper ) possess tryptophan residue (W) instead of aspartic acid residue. Other R genes with LRR3 super family domain reported earlier were NBARC domain of putative rp3 protein from Zea mays, RPP5 disease resistance protein of A. thaliana and NBARC domain of R genes of Solanaceae, O. sativa, Rosids, Vitis vinifera (RX-CC-NBARC), Malus domestica, Capsicum annuum and Citrus sp. LRR domain of known R genes (rp3, RPP5, NBARC) was present in IIHRR13-4 RGAs. The presence of conserved domain LRR 3 super family will give unique identity to RGAs identified from the genome of powdery mildew resistance germplasm line IIHRR13-4. Several RGAs present in each plant genome may or may not link to resistance. The IIHRR13-4 was found resistant to powdery mildew in field evaluations. The J. Hortl. Sci. Vol. 15(1) : 81-92, 2020 Resistance gene analogues (RGA) in rose 90 powdery mildew resistant line IIHRR13-4 was studied along with other resistant and susceptible rose lines. The RGAs were identified from all rose genotypes and some were excluded because of stop codons. Finally eleven RGAs selected from the IIHRR13-4 that might be linked to resistance mechanisms. This is the initial study on resistance mechanisms in the rose line IIHRR13-4 against powdery mildew disease. The expression analysis studies (N. K Chandran, Personal communication) revealed more expression of RGAs and resistance related transcription factors in IIHRR13-4 compared to susceptible cultivar konfetti upon powder y mildew infection. T he comparison between IIHRR13-4 and konfetti revealed that expression level of RGA transcripts might not be sufficient to elicit resistance in konfetti. This indicates the importance of proper and required expression of disease resistance gene against particular pathogen. This study indicates that several R gene candidates (RGAs) are present in rose plants but only few are linked to disease resistance. These RGAs identified from IIHRR13-4 might be putative derivatives for R gene(s) against powdery mildew and may help in future research on mapping and characterization of R genes from IIHRR13-4. Map based cloning approach is used to isolate R genes and that requires high-density genetic maps. Genome-wide RGA identification would assist to develop markers and mapping resistance genes and further possible cloning. CONCLUSION The present study explains the putative molecular mechanism behind resistance to powdery mildew resistance in IIHRR13-4 through different motifs present in the NBS domain of NBS-LRR group of R genes. This can be used as a basis for further studies related to molecular mechanism of resistance since RGAs a r e potentia l ca ndida tes for functiona l resistance gene and marker development in various breeding programs. The results of present study will help to develop RGA based markers linked to powdery mildew resistance in rose and this will help in rose resistant breeding and disease resistance screening programs using R gene profiling. Further study related to expression level of RGAs will provide more insight into molecular basis of disease resistance. ACKNOWLED’GEMENT The authors thank the Director, ICAR-IIHR and Head, Division of Crop Protection, ICAR-IIHR, Bangalore for providing the facilities to conduct the research work. Chandran et al. J. Hortl. Sci. Vol. 15(1) : 81-92, 2020 REFERENCES Aarts, M. G., Hekkert, B. L., Holub, E. B., Beynon, J. L, Stiekema, W. J. and Pereira, A. 1998. Identification of R-gene homologues DNA fr a gments genetica lly linked to disea se resistance loci in Arabidopsis thaliana. Mol. Plant. Microbe. Interact. 11: 251-258. Ayana, T. D., Yasmin, A., Le, L. T., Kaufmann, H., Biber, A. and Kuhr, A. 2011. Mining disease resista nce genes in roses: functional a nd molecular characterization of the Rdr1 locus. Front. Plant Sci. 2:1-11. Belkhadir, Y., Subramaniam, R. and Dangl, L.G. 2004. Plant disease resistance protein signaling: NBS– LRR proteins and their partners. Curr. Opin. Plant Biol.7: 391–399. Biber, A., Kaufmann, H., Linde, M., Spiller, M., Terefe, D. and Debener, T. 2010. Molecular markers from a BAC contig spanning the Rdr1 locus: A tool for marker assisted selection in roses. Theor. Appl. Genet. 120: 765-773. Calenge, F., van der Linden, C. G., van de Weg, E., Schouten, H. J., Van Arkel, G., Denance, C. a nd Dur el, C. E. 2005. Resista nce gene analogues identified through the NBS profiling method map close to major genes and QTL for disea se resistance in apple. Theor. Appl. Genet. 110: 660–668. Collier, M.S. and Moffett, P. 2009. NB-LRRs work a ‘‘bait and switch’’ on pathogens. Trends. Plant Sci. 14:521-529. Dangl, J. L. and Jones, J.D.G. 2001. Plant pathogens and integrated defense responses to infection. Nature. 411: 826–833. Debener, T. and Byrne, H.T. 2014. Disease resistance breeding in rose: current status and potential of biotechnological tools. Plant Sci. 228:107-117. 91 J. Hortl. Sci. Vol. 15(1) : 81-92, 2020 Resistance gene analogues (RGA) in rose Deng, Z., Huang, S., Ling, P., Chen, C., Yu, C., Weber, C., Moore, G.A. and Gmitter, Jr. F.G. 2000. Cloning and characterization of NBS-LRR class resistance-gene candidate sequences in citrus. Theor. Appl. Genet. 101: 814-822. Dettori, M.T., Quarta, R. And Verde, I. 2001. A peach linkage map integrating RFLPs, SSRs, RAPDs, a nd mor p h ol og ic a l ma r ke r s . G e n o m e . 44:783-790. Dirlewanger, E., Graziano, E., Joobeur, T., Garriga- Ca ldere, F., Cosson, P., Howa d, W. And Arus, P. 2004. Comparative mapping and marker assisted selection in Rosaceae fruit crops. Proc. Natl. Acad Sci. 101:9891– 9896. Dirlewanger, E., Pascal, T., Zuger, C. and Kervella, J. 1996, Ana lysis of molecula r ma r ker s associated with powdery mildew resistance genes in p ea c h [ ( Pr u n u s p e r s i c a (L. )Batsch)] Prunus davidiana hybr ids. Theor. Appl. Genet. 93:909–919. Doyle, J.J. and Doyle, J.L A. 1987. Rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin. 19:11-15. Ellis, J. and Jones, D. 1998. Structure and function of p r ot ein s c ont r olling s t r a i n- s p ec if ic pathogen resistance in plants. Curr. Opin. Plant Biol. 1:288-293. Ellis, J., Dodds, P. and Pryor, T. 2000. Structure, function a nd evolution of p la nt disea se resistance genes. Curr. Opin. Plant Biol. 3: 278-284. Ellis, J.G. and Jones, D.A. 2003. Plant disease r es is t a nc e genes . I nna t e I mmu nit y. Infectious Disease (eds.) Ezekowitz R.A.B. and Hoffmann J.A.), Human Press, Totowa, NJ. pp. 27-45. Flor, H.H. 1971. Current status of the gene-for- gene concept. Annu. Rev. Phytopathol. 9: 278–296. Hall, T.A. 1999. BioEdit: a user-friendly biological sequence a lignment editor a nd a na lys is p r ogr a m f or Windows 95 /9 8/ NT. Nu cl. Acids. Symposium Series. 41:95-98. Hammond-Kosack, K. and Jones, J.D.G. 1997. Plant disease resistance genes. Annu. Rev. Plant Physiol. Plant. Mol Biol. 48: 575– 607. Hammond-Kosack, K.E., Jones, D.A. and Jones, J . D . G. 1 9 9 6 . E ns na r ing mic r ob es : t he components of plant disease resistance. New Phytol. 133 :11-24. Hattendorf, A.andDebener, T. 2007. Molecular characterization of NBS-LRR-RGAs in the rose genome. Physiol. Plant. 129 : 775-786. Hattendorf, A.andDebener, T.2007. NBS-LRR-RGAs in roses: diversity, genomic organiza tion, expression and chromosomal location. Acta Hortic. 751:151-162. Hattendorf, A., Linde, M. and Mattiesch, L.2004. Genetic analysis of rose resistance genes and their localization in the rose genome, Acta Hortic. 651:123-130. Holt, III. F.B., Mackey, D.AndDangl, L.J.2000. Recognition of pathogens by plants. Curr. Biol. 10: R5-R7. Jones, D.G.J.2001. Putting knowledge of plant disease resistance genes to work. Curr.Opin. Plant. Biol. 4: 281-287. Lalli, D.A., Decroocq, V., Blenda, A.V., Schurdi- Levraud, V., Garay, L., Le, G. O., Damsteegt, V. , Reighard, G.L. and Abbott, A.G. 2005. Identification and mapping of resistance gene analogs (RGAs) in Prunus: a resistance map for Prunus. Theor. Appl. Genet. 111:1504– 1513. Leus, L. , Huylenbr oeck, V. J. , Bocksta ele, V. E. a ndHofte, M. 2003. Bioa ssa ys for r esistance scr eening in commer cia l r ose breeding. Acta Hortic. 651:39-45. Linde,M. a nd Debener, T. 2003. Isola tion a nd identification of eight races of powdery mildew of roses (Podosphaerapannosa(Wallr.:Fr.) de Bary) and the genetic analysis of the resistance gene Rpp1. Theor. Appl. Genet. 107: 256-62. Linde, M., Mattiesch, L.and Debener, T.2004. Rpp1, a domina nt gene providing r a ce-specific r esista nce to r ose powder y mildew (Podosphaera pannosa): molecular mapping, 92 Chandran et al. J. Hortl. Sci. Vol. 15(1) : 81-92, 2020 (Received on 17.01.2020 and accepted on 18.03.2020) SCAR development and confirmation of disease resistance data. Theor. Appl. Genet. 109: 1261-1266. McHale, L., Tan, X., Koehl, P. and Michelmore, R.W. 2006. Plant NBS-LRR proteins: adaptable guards. Genome Biology. 7:212. Meyers, B.C., Dickerman, A.W., Michelmore, R.W., Sivaramakrishnan, S., Sobral, B.W. and Young, N.D. 1999. Plant disease resistance genes encode members of an ancient and diverse protein family within the nucleotide-binding super-family. Mol. Plant. Microbe Interact. 20:317–332. Meyers, B.C., Kozik, A., Griego, A., Kuang, H. And Michelmore, R.W. 2003. Genome-wide analysis of NBS-LRR-encoding genes in Arabidopsis. Plant Cell. 15: 809–834. Perazzolli, M., Malacarne, G., Baldo, A., Righetti, L., Bailey, A., Fontana, P., Velasco, R. and Malnoy, M. 2014. Characterization of resistance gene a na logues (RGAs) in Apple (Malus X domestica Borkh.) and their evolutionary history of the Rosaceae Family. Plos One. 9 : 2. e83844. Pessina, S., Pavan, S., Catalano, D., Gallotta, A., Visser, R., Bai, Y., Malnoy, M. and Schouten, H. 2014. Characterization of the MLO gene family in Rosa cea e a nd gene expression a na lysis in Malus domestica. BMC Genomics. 15: 618. Phukan, U.J., Jeena, G.S., and Shukla, R.K. 2016. WRKY Transcription Factors: Molecular regulation and stress responses in plants. Front. Plant Sci. 7:760. Quarta, R., Dettori, M.T., Sartori, A. and Verde, I. 2000. Genetic linkage map and QTL analysis in peach. Acta Hortic. 521:233–238. Saraste, M., Sibbald, P.R. and Wittinghofer, A. 1990. The P-loop, a common motif in ATP-and GTP- binding pr oteins. Trends. Biochem. Sci. 15:430-434. Sekhwal, K.M., Li, P., Lam, I., Wang, X., Cloutier, S. and You, M.F. 2015. Disease resistance gene analogs (RGAs) in plants. Int. J. Mol. Sci. 16:19248-19290. Song, W.Y., Pi, L.Y., Wang, G.L., Gardner, J., Holsten, T.and Ronald, P.C. 1997. Evolution of the rice Xa21 disease resistance gene family. The Plant Cell. 9:1279-1287. Tamura, K., Stecher, G., Peterson, D., Filipski, A. and Kuma , S. 2013. MEGA6 : Molecula r Evolutionary Genetics Analysis version 6.0. Mo. Biol. Evol.  30:2725-2729. Von Malek, B., Weber, W.E. and Debener, T., 2000. Identification of molecular markers linked to Rdr1, a gene conferring resistance to blackspot in roses. Theor. Appl. Genet. 101:977-983. Vossen, H.J., Dezhsetan, S., Esselink, D., Arens, M., Sanz, J. M. And Verweij, W. 2013. Novel applications of motif-directed profiling to identify disease resistance genes in plants. Plant Methods. 9:37. Xu, Q., Wen, X. and Deng, X. 2005. Isolation of TIR a nd non TIR NBS-LRR r esista nce gene analogues and identification of molecular markers linked to a powdery mildew resistance locus in chestnut rose (Rosa roxburghii Tratt). Theor. Appl. Genet. 111:819-830. Xu, Q., Wen, X. and Deng, X. 2007. Phylogenetic and evolutionary analysis of NBS-encoding genes in Rosaceae fruit crops. Mol. Phylogenet. Evol. 44:315–324. Young, N.D. 2000. The genetic architecture of resistance. Curr. Opin. Plant. Biol. 3:285–290. Yu, Y.G., Buss, G.R. and Maroof, M. 1996. Isolation of a super -fa mily of ca ndida te disea se r esista nce genes in soybea n ba sed on a conserved nucleotide-binding site. Proc. Natl. Acad sci. 93: 11751-11756. Zamora, M.M., Castagnaro, A. and Ricci, J.D. 2004. Isolation and diversity analysis of resistance gene analogues (RGAs) from cultivated and wild strawberries. Mol. Genet and Genomics. 272 : 480-487.