Isolation, Characterization and Sequence Analysis of Resistance Gene Analogs (RGAs) in Plants Related to Huanglongbing(HLB)

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1 Isolation, Characterization and Sequence Analysis of Resistance Gene Analogs (RGAs) in Plants Related to Huanglongbing(HLB) Liu Tingting, Yin Youping, Lin Yayu, and Wang Zhongkang Key Laboratory of Gene Function and Regulation at Chongqing, College of Bioengineering at Chongqing University, Chongqing , China Abstract. Citrus production is seriously affected by HLB to which no resistant plant is available in the cultivated germplasm. Degenerate primers designed based on conserved NBS (nucleotide binding site) motifs contained in some plant resistance genes were used to isolate analogous sequences called resistance gene analogs (RGAs). About xx resistance gene analogs (RGAs) were cloned from several spices related to HLB using PCR method with primers above-mentioned. The BLASTN searches revealed that 43 DNA fragments showed different sequence homology with known plant R-genes or deposited RGAs and 12 sequences could be translated to polypeptides without stop codons. These RGAs and 12 deduced amino acid were analyzed by Clustalx and DNAMAN software. Sequence analysis showed these RGAs contain the conserved domains P-loop, Kinase-2a, Kinase-3a, and HD, which was conserved in NBS-LRR type disease resistance gene. These 12 RGAs shared 37.39%-43.43% identity with the resistance genes of TobaccoNÂFlaxL6ÂArabidopsis thaliana RPS2ÂRPS5Â RPP8ÂRPM1. Gene duplication followed by sequence divergence were proposed as the mode for the evolution of a large number of distantly or closely related RGA genes in plants, and this mode may play a role in the generation of new resistance specificity and cloning related resistance genes about HLB. Keyword: Huanglongbing(HLB), Resistance gene analogs(rgas), NBS conserved region, Degenerate primers. 1 Introduction Plants defend themselves against pathogens in many sophisticated mechanisms, one of which is the gene-for-gene hypothesis, where resistance (R) genes perform an important role in defense-response initiation [1]. Numerous disease R genes have been cloned and characterized from plenty of plant species either by map-based cloning [2] or transposon tagging [3] since the first R gene Hml was cloned. Comparative analysis of these R-genes have shown some conserved amino acid including nucleotide-binding sites (NBS), leucine-rich repeat (LRR), serine/threonine protein kinase (PK) and transmembrane domains (TM) [1]. The largest known R genes so far encode a nucleotide-binding site (NBS) and leucine-rich repeats (LRR) domain. The NBS E. Zhu and S. Sambath (Eds.): Information Tech. and Agricultural Eng., AISC 134, pp springerlink.com Springer-Verlag Berlin Heidelberg 2012

2 836 T. Liu et al. regions of R genes cloned contain several highly conserved motifs such as P-loop, kinase-2, kinase-3a, and GLPL, etc. The P-loop and kinase-2 are responsible for ATP/GTP binding and hydrolysis in defense system [4]. The Kinase-3a and GLPL are part of a putative weak hydrophobic region in R genes. Using PCR method with degenerate primers according to the conserved motifs, a large number of NBS-encoding sequences homology to R genes, so-called R gene analogs (RGAs), have been successfully obtained from many kinds of plant species such as rice[5]â tabacco[6] potato[7]. Actually, RGAs are rich in plant genome, which provides a facile system to study the evolutionary biology and genetics of NBS-encoding gene family [8]. Huanglongbing (HLB) is one of the most destructive diseases of citrus [9]. HLB affects not only all known citrus species and citrus relatives with little known resistance but other plants. Sweet oranges, mandarins and tangelos were found to be highly susceptible to HLB. Some previous research and field observations of our laboratory revealed that sour orange, grapefruits, murraya paniculata and some commercially important citrus varieties show a little different resistance to HLB [10. 11]. Most of these species are woody perennials with a long generation time and big plant canopy, which makes the classic genetic analysis more difficult and therefore lead to their genomes hardly known [12]. More and more researchers come to realize the importance of molecular genetics. It is reported that tree fruit Genome Database Resources (tfgdr), the Citrus Genome Database will house the genomics, genetics and breeding data for major citrus crops such as orange, grapefruit, mandarin, tangerine, lemon, lime and pummelo. Genome information governs important traits and so will have great potential use in plant breeding. In this study, using PCR-based strategy with degenerate primers, 43 RGAs were isolated from several plants genome related to HLB. At the same time, we download 14 other RGAs form NCBI. A total of 59 RGAs were collected and used for evolutionary analysis. The phylogeny was comparatively analyzed both on species and genus level, suggesting that gene duplication followed by divergence may have occurred after the radiation of species or genus. 2 Materials and Methods 2.1 Primers and PCR Amplification of RGA Sequences Five degenerate primers were used in six combinations (Table 1) for PCR amplifications. Primer F1 was based on the sequence GGVGKTT the amino-acid sequence found in the P-loop of N, L6 and RPS2; primers R1, R2 and R3 were designed according to the sequence GLPLAL. Total genomic DNA was extracted from leaves of Citrus maxima and Murraya paniculata. PCR reaction was carried out in a volume of 25 µlµl containing 5 U μl -1 Taq DNA polymerase (TAKARA, Japan), 10 PCR buffer, 25 mm L -1 MgCl 2, 2.5 mm L -1 dntp, 10 µm of each primer and 20 ng μl -1 template DNA. Touchdown-PCR amplification was performed for an initial denaturation at 94 C for 5 min, followed by 20 amplification cycles of 94 C for 30 s, at 0.5 C of C for 30 s and 72 C for 30s, and then 30 cycles of 94 C for 30 s, 45 C for 30 s and 72 C for 30s, a final extension step at 72 C for 7 min. The PCR reaction with deionized water as template was used for a negative control.

3 Isolation, Characterization and Sequence Analysis of RGAs 837 Table 1. The primers used for the PCR amplification of RGAs Prime Prime sequence 5-3 Conserved amino acid name cquf1 GGDGTDGGNAARACWAC GGVGKTT(P-loop) cquf2 GGIGGIRTIGGNAAIACIAC (L/I/V)LVLDDVW cqur1 AGIGCHAGNGGNAGNC GLPLAL cqur2 AGNGCHAGNGGYAANCC GLPLAL cqur3 AANGCHAGNGGYAANCC GLPLAL Codes for degenerate positions: W = A/T; R = A/G; H = A/T/C; N = A/T/G/C; Y = C/T; D = A/G/T; V = G/A/C; I = inosine; F: forward orientation; R: reverse orientation. 2.2 RGA Cloning and Restriction Analysis PCR products were separated on a 1.5% agarose gel, and DNA fragments equal or larger than the expected sizes were recovered for cloning. The purified PCR products were cloned into the plasmid pmd-19 simple vector (TaKaRa, Japan). Plasmid DNA was isolated from randomly selected positive clones and digested overnight at 37 o C with RsD, HaH and MsS restriction enzymes (Promega, USA). Fragments were separated by electrophoresis on a 2% agarose gel. According to the restriction profiles, clones with a unique restriction profile were selected for sequencing. 2.3 Sequences Analysis Phylogenetic analysis was executed to evaluate further relationship among these RGAs and plant R genes. Multiple alignments of nucleotide and deduced amino acid sequences were performed using ClustalX [13] and DNAMAN software, and then the output was edited by the GeneDoc software [14]. A phylogenetic tree was constructed with the MEGA4 software. The trees were visualized using the program TREEVIEW. 3 Results and Analysis 3.1 Amplification and Collection of RGA Sequences Seventy-nine NBS sequences were collected for identification by the below methods: 14 published sequences form NCBI; and 43 sequences isolated by PCR amplifying with degenerate primers (Table 1), fragments of target sizes were amplified from Citrus maxima and Murraya paniculata. The approximately 500 bp band from each primer combination was close to the fragment size expected and therefore these bands were cloned. Restriction analyses showing different restriction patterns of insert fragments were identified and characterized further by sequencing and sequence analysis. 3.2 Sequence and Phylogenetic Analysis A total of 43 clones which contains 15 from Citrus maxima (accession NO.: HM HM800480), 28 from Murraya paniculata (accession NO.: HM HM769650) were obtained. The BLASTN searches revealed that 43 DNA

to resistance genes. 12 sequences could be translated to polypeptides without stop codons, and they showed strong overall similarities to several plant R-gene sequences.

4 838 T. Liu et al. fragments showed different sequence homology with known plant R-genes or deposited RGAs, which one clones were highly similar to Helianthus arqophyllus gene, some had no or only very weak similarity to resistance genes. 12 sequences could be translated to polypeptides without stop codons, and they showed strong overall similarities to several plant R-gene sequences. Multiple alignments performed with these RGAs and the three most similar R-gene peptide sequences showed that the similarity was especially high at the three NBS motifs (P-loop, kinase-2, and kinase-3a) (Fig. 1). Fig. 1. Alignment of the deduced amino acids of 12 RGA sequences with the NBS domain of eight R genes from the GenBank database using DNAMAN [11]: Tobacco N (U15605), Flax L6 U27081), Arabidopsis thaliana RPS2 (U14158), RPM1 (AAF27008), RPP8 (AF089710), and RPS5 (AF074916),Tomato I2C (AF004878),CTV4(AAN ). Conserved amino acids such as the P-loop, Kinase - 2, GLPL motifs are highlighted. 57 sequences include 15 from Citrus maxima, 28 from Murraya paniculata and 14 from NCBI was used for phylogenetic trees construction. The deduced amino acid sequences of the 12 RGAs and 3 known NBS-LRR resistance proteins from other plant species were pooled for a protein phylogenetic analysis. Tree construction and bootstrap tests were carried out as described previously. The result of neighbor-joining phylogenetic tree was shown in Fig. 2.

5 Isolation, Characterization and Sequence Analysis of RGAs 839 Fig. 2. Phylogenetic comparison of Citrus maxima, Murraya paniculata RGAs. Sequences were aligned using ClustalX, and the tree was generated using the neighbor-joining method. Bootstrap values (1000 replicates) with only values >50% were shown on the branches. 4 Discussion RGAs have been successfully isolated from many plants using PCR amplification with degenerate primers based on the conserved motifs of known plant R-genes. While no molecular cloning of putative R genes from plants related to HLB has yet been reported, the objective of this study is to isolate and characterize RGAs from plants related to HLB and to evaluate their diversity and phylogenetic relationships. The relation of RGAs to plant R-genes may have three categories: (1) RGAs are actual R-genes. For example, the full-length cdna of the soybean R-gene KR1 was cloned using an RGA fragment as a hybridization probe to screen a cdna library [15]; (2) RGAs are linked to plant R-genes, for instance, NBS-encoding RGAs of sunflower are linked to down mildew-resistant locus P15/P18 [16], and three RGA sequences were shown to be co-segregated with wheat rust resistance gene [17]; (3) RGAs are not functionally related to R-genes. This result may be depended on our choice to the primers or the limited in number of RGAs sequenced as well.

6 840 T. Liu et al. Meyers et al. [18] analyzed the NBS domains of the plant NBS-LRR class R genes and a large number of RGAs and found that they could be classified into either TIR (Toll/Interleukin-1 receptor homology) or non-tir groups. In their analysis, N and L6 belong to the TIR group, while RPS2, RPM1 and I2C-2 fall into the non-tir group. Our data indicate that J2, J19 and J14 formed a major cluster with N and L6, and they have an aspartic acid residue (D or N) at the final residue position of the kinase-2 motif that is often seen in the TIR group. Most of citrus RGAs were clustered with RPS2 and have a tryptophan residue (W), indicating that they might belong to the non-tir group. Attempts are being made to obtain the upstream sequence information; when this becomes available, it should assist in the classification of these citrus RGAs regarding TIR or non-tir grouping. Most cloned plant R-genes encode proteins with the NBS region, which is required for ATP or GTP binding. However, genes involved in development process and other signal transduction pathways not relevant to plant disease resistance also encode proteins containing a conserved NBS region [19]. Likewise, apart from their role in plant disease resistance, genes encoding kinases also function in other aspects of plant physiology. The isolation of RGAs will provide valuable resources for further clarifying the molecular mechanism of HLB resistance. References 1. Dangl, J.L., Jones, J.D.G.: Plant pathogens and integrated defence responses to infection. Nature 411, (2001) 2. Dixon, M.S., Hatzixanthis, K., Jones, D.A., Harrison, K., Jones, J.D.G.: The tomato Cf-5 disease resistance gene and six homologs show pronounced allelic variation in leucine-rich repeat copy number. Plant Cell 10, (1998) 3. Anderson, P., Lawrence, G., Morrish, B., Aylife, M., Finnegan, E., Ellis, J.: Inactivation of the Xax rust resistance gene M associated with the loss of a repeated unit within the leucine-rich repeat coding region. Plant Cell 9, (1997) 4. Belkhadir, Y., Subramaniam, R., Dangl, J.L.: Plant disease resistance protein signaling: NBS-LRR proteins and their partners. Curr. Opin. Plant Biol. 7, (2004) 5. Zheng, X., Cui, W., Li, X.: NBS-LRR disease resistance analogues of Rice Varieties. Science in China 31(1), (2001) 6. Gao, Y., Xu, Z., Jiao, F., Yu, H., Xiao, B., Li, Y., Lu, X.: Cloning, structural features, and expression analysis of resistance gene analogs in Tobacco. Mol. Biol. Rep. 37, (2010) 7. Brugmans, B., Wouters, D., van Os, H., et al.: Genetic mapping and transcription analyses of resistance gene loci in potato using NBS profiling. Theor. Appl. Genet. 117, (2008) 8. Meyers, B.C., Dickerman, A.W., Michelmore, R.W., Sivaramakrishnan, S., Sobral, B.W., Young, N.D.: Plant disease resistance genes encode members of an ancient and diverse protein family within the nucleotide-binding superfamily. Plant J. 20, (1999) 9. Bove, J.M., Huang, L.: A destructive, newly-emerging, century-old disease of citrus. Plant Pathol. 88(1), 7 37 (2006) 10. Nariani, T.K.: Integrated approach to control citrus greening disease in India. Proc. Int. Soc. Citricult. 1, (1981)

7 Isolation, Characterization and Sequence Analysis of RGAs Miyakawa, T.: Experimentally-induced symptoms and host range of citrus likubin (greening disease). Annu. Phytopathol. Soc. Jpn. 46, (1980) 12. Dirlewanger, E., Graziano, E., Joobeur, T., Garriga-Caldere, F., Cosson, P., Howad, W., Arus, P.: Comparative mapping and markerassisted selection in Rosaceae fruit crops. Proc. Natl. Acad. Sci. 101, (2004) 13. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G.: The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25, (1997) 14. Nicholas, K.B., Nicholas Jr., H.B., Deerfield II, D.W.: GeneDoc: analysis and visualization of genetic variation. EMBnet News 4, (1997) 15. He, C.Y., Tian, A.G., Zhang, J.S., Zhang, Z.Y., Gai, J.Y., Chen, S.Y.: Isolation and characterization of a full-length resistance gene homolog from soybean. Theor. Appl. Genet. 106, (2003) 16. Radwan, O., Bouzidi, M.F., Vear, F., Philippon, J., De Labrouhe, D.T., Nicolas, P., et al.: Identification of non-tir-nbs-lrr markers linked to the Pl5/Pl8 locus for resistance to downy mildew in sunflower. Theor. Appl. Genet. 106, (2003) 17. Yan, G.P., Chen, X.M., Line, R.: Resistance gene-analog polymorphism markers co-segregating with the Yr5 gene for resistance to wheat stripe rust. Theor. Appl. Genet. 106, (2003) 18. Meyers, B.C., Dickerman, A.W., Michelmore, R.W., Sivaramakrishnan, S., Sobral, B.W., Young, N.D.: Plant disease resistance genes encode members of an ancient and diverse protein family within the nucleotide-binding superfamily. Plant J. 20, (1999) 19. Shirasu, K., Schulze-Lefert, P.: Regulators of cell death in disease resistance. Plant Mol. Biol. 44, (2003)

ABSTRACT. tropical and sub-tropical countries. Recent utility of turmeric by the pharmaceutical

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