Funct Integr Genomics (2015) 15:233–245 DOI 10.1007/s10142-014-0416-x
ORIGINAL PAPER
Stage-specific reprogramming of gene expression characterizes Lr48-mediated adult plant leaf rust resistance in wheat Raman Dhariwal & Vijay Gahlaut & Bhaganagare R. Govindraj & Dharmendra Singh & Saloni Mathur & Shailendra Vyas & Rajib Bandopadhyay & Jitendra Paul Khurana & Akhilesh Kumar Tyagi & Kumble Vinod Prabhu & Kunal Mukhopadhyay & Harindra Singh Balyan & Pushpendra Kumar Gupta
Received: 11 June 2014 / Revised: 9 November 2014 / Accepted: 17 November 2014 / Published online: 29 November 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Wheat genotype CSP44 carrying a recessive gene Lr48 exhibits adult plant resistance (APR; incompatible reaction) but gives a compatible reaction (susceptibility) at the seedling stage against leaf rust. A comparative gene expression analysis involving cDNA-amplified fragment length polymorphism (cDNA-AFLP) and quantitative PCR (qPCR) was carried out for incompatible and compatible reactions in the genotype CSP44. cDNA-AFLP analysis was conducted using RNA samples that were isolated from flag leaves following inoculation with leaf rust race 77–5 (the most virulent race) and also after mock inoculation. As many as 298 of a total of 493 expressed transcript-derived fragments Electronic supplementary material The online version of this article (doi:10.1007/s10142-014-0416-x) contains supplementary material, which is available to authorized users. R. Dhariwal : V. Gahlaut : H. S. Balyan : P. K. Gupta (*) Molecular Biology Laboratory, Department of Genetics and Plant Breeding, Ch. Charan Singh University, Meerut, India e-mail:
[email protected] B. R. Govindraj : K. V. Prabhu Division of Genetics, Indian Agricultural Research Institute, New Delhi, India D. Singh : R. Bandopadhyay : K. Mukhopadhyay Department of Bio-Engineering, Birla Institute of Technology, Mesra, India S. Mathur : S. Vyas : J. P. Khurana Interdisciplinary Centre for Plant Genomics and Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi, India A. K. Tyagi National Institute of Plant Genome Research, New Delhi, India R. Dhariwal Agriculture and Agri-Food Canada, Lethbridge Research Centre, Lethbridge, Canada
(TDFs) exhibited differential expression (262 upregulated and 36 downregulated). Of these 298 TDFs, 48 TDFs were eluted from gels, re-amplified, cloned, and sequenced. Forty two of these 48 TDFs had homology with known genes involved in the following biological processes: energy production, metabolism, transport, signaling, defense response, plant-pathogen interaction, transcriptional regulation, translation, and proteolysis. The functions of the remaining six TDFs could not be determined; apparently, these represented some novel genes. The qPCR analysis for 18 TDFs (with known and unknown functions, but showing major differences in expression) was conducted using RNA isolated from the seedlings as well as from the adult plants. The expression of at least 11 TDFs was induced and that of 4 other TDFs attenuated or remained near normal in adult plants following leaf rust inoculations. The remaining three TDFs had nonspecific/developmental stage-specific expression. Functional annotation of TDFs that were upregulated suggest that the APR was supported by transient recruitment and reprogramming of processes like perception and recognition of pathogen effector by receptors, followed by CDPK and MAPK signaling, transport, metabolism, and energy release. Keywords Triticum aestivum . Puccinia triticina . TDFs . Incompatible interaction . Compatible interaction . Recessive APR gene
Introduction Wheat (Triticum aestivum L.) crop suffers severe losses in yield worldwide due to leaf rust caused by Puccinia triticina Eriks.; these losses sometimes can be as high as 65 % (Saari and Prescott 1985). These losses can be minimized through the use of Lr genes for leaf rust resistance. As many as 73 Lr
234
genes are now known for leaf rust resistance (Park et al. 2014); majority of these genes provide seedling resistance (SR), which is often short-lived, since new races of the leaf rust pathogen emerge quickly and overcome Lr genes imparting SR (Singh et al. 2013). A number of genes imparting adult plant resistance (APR) are also known and include the following: Lr12, Lr13, Lr22a, Lr22b, Lr34, Lr46, Lr48, Lr49, and Lr67. The Lr genes for APR are often used in combination with Lr genes for SR, for developing wheat cultivars with durable resistance. At least three genes imparting SR (Lr1, Lr10, and Lr21) and one gene imparting APR (Lr34) have also been cloned and characterized (Feuillet et al. 2003; Huang et al. 2003; Cloutier et al. 2007; Krattinger et al. 2009), suggesting that genes for SR encode proteins with nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domains, while APR gene Lr34 belongs to an ABC transporter gene family. In higher plants, the resistance against a pathogen is governed at two levels involving two different signal transduction pathways, one involving pathogen-associated molecular patterns (PAMP)-triggered immunity (PTI) providing basal resistance, and the other involving effector-triggered immunity (ETI) providing gene-specific defense (Jones and Dangl 2006). Following pathogen challenge, and depending on the R gene involved, resistant plants respond rapidly through production of novel transcripts belonging to various pathways involved in metabolism and defense (Torres et al. 2003; Bolton et al. 2008; Giraud et al. 2012). The defense responses, in turn, are also associated with a complex network of downstream signaling pathways that involve the following three signaling molecules: salicylic acid, jasmonic acid, and ethylene (Dong 1998; Thomma et al. 2001; Kunkel and Brooks 2002; Nandi et al. 2003; Hua 2009; Giraud et al. 2012; Mur et al. 2013; Van der Does et al. 2013). It is also known that the abundance of new transcripts and deployment time of signaling pathways are stress-dependent (Torres et al. 2003). Keeping the above in view, gene expression studies involving R genes have been conducted in many host-pathogen systems including leaf rust pathosystem in wheat. The approaches used for this purpose included cDNA-amplified fragment length polymorphism (cDNA-AFLP), microarray analyses, and development of ESTs (Zhang et al. 2003; Fofana et al. 2007; Hulbert et al. 2007; Bolton et al. 2008; Manickavelu et al. 2010; Dhariwal et al. 2011; Spielmeyer et al. 2013; Kumar et al. 2014). However, not many gene expression studies involving APR Lr genes in wheat have been conducted; in particular, gene Lr48 has never been studied in this manner. Wheat genotype CSP44 carries the APR gene Lr48, which is recessive in nature, and is located on chromosome arm 2BL (Saini et al. 2002; Khanna et al. 2005; Singh et al. 2011). This APR gene is responsible for a hypersensitive response (HR;
Funct Integr Genomics (2015) 15:233–245
infection type 0) to leaf rust (race 77-5) infection at the adult plant (booting) stage, and imparts resistance against most leaf rust pathotypes prevalent in India; some other APR genes like Lr34 and Lr46 lack hypersensitive response (Bansal et al. 2008). The resistance provided by Lr48 gene at adult plant (booting) stage is comparable to the resistance provided by any dominant gene for seedling resistance. However, at the seedling stage, this gene fails to provide resistance, so that the crop at the seedling stage is susceptible to leaf rust despite the presence of Lr48. No information is available on the molecular mechanism involved in Lr48-mediated APR that follows a susceptible reaction at the seedling stage. Keeping the above in view, we conducted a gene expression study in wheat cultivar CSP44; the study included cDNA-AFLP, qPCR, and bioinformatics analyses. A number of genes were identified which were downregulated during the course of compatible interaction (susceptible response) at the seedling stage, but were upregulated during incompatible interaction (resistance response) at the adult plant stage, following inoculation of the host genotype by the most virulent pathogen race 77-5.
Materials and methods Plant material Wheat genotype CSP44 was used for transcription profiling. This stock was derived from a single plant selection from the Australian cultivar “Condor” (Saini et al. 2002). Pathogen race Single spore-derived inoculum of one of the most prevalent and virulent pathogen race 77-5 (syn. 121R63-1) of P. triticina was procured from the Regional Station, Directorate of Wheat Research (DWR), Flowerdale, Shimla. This race exhibits virulence at seedling stage and avirulence at adult plant stage against the APR gene Lr48 and was used for inoculating the seedlings and the flag leaves of the host genotype CSP44. Plant growth conditions CSP44 plants were raised in growth chamber under controlled conditions of 16 h day (25 °C; with 450 μmol m-2 s-1 light intensity) and 8 h night (18 °C) at the National Phytotron Facility, Indian Agricultural Research Institute, New Delhi. Experimental design Independent experiments were designed for cDNA-AFLP analysis and qPCR analysis, since results of cDNA-AFLP
Funct Integr Genomics (2015) 15:233–245
analysis were subsequently utilized for planning qPCR experiments. cDNA-AFLP analysis Flag leaves of CSP44 plants at boot leaf stage [Zadoks scale (Z) 49; Zadok et al. 1974] were inoculated and samples collected for isolation of RNA to be used for cDNA-AFLP analysis (seedlings were not used for cDNA-AFLP analysis). Fungus inoculations were performed by brushing urediniospores (of leaf rust race 77-5) suspension [in water fortified with Tween-20® (0.75 μl/ml) at an average concentration of 20 urediniospores/microscopic field (x10 Χ x10; 1.8 mm)]; these plants provided samples for treatments. Similarly, mock inoculations were performed using suspension without urediniospores, and were used as controls. These plants were incubated for 36 h in a humid chamber (RH ≥95 %) maintained at 25 °C (16 h)/18 °C (8 h) temperature in dark, after which normal conditions were restored. In each case, leaf samples were collected from two independent biological replicates at each of the following durations after inoculation: 0 h (Z49), 24 h (Z50), 48 h (Z53), 72 h (Z55), 96 h (Z57), and 168 h (Z60) (the durations were selected on the basis of Zadoks scale described by Zadok et al. 1974). The time-points were selected from the following three generic phases of host response (HR): 0–24 h (phase I: perception and recognition of pathogen), 24–96 h (phase II: transduction and early expression of plant defense), and 96–168 h (phase III: expression of plant defense and induction of HR). Inoculated CSP44 adult plants were also examined regularly on a daily basis during the above three generic phases and phenotypic symptoms of resistance were recorded (Supplementary Fig. 1). qPCR analysis Leaf samples for qPCR were collected both from seedlings and adult plants at the same six time-points after inoculation, following the procedure that was followed for cDNA-AFLP analysis. For qPCR analysis, samples collected from two independent biological replicates of pathogen-inoculated and mock-inoculated seedlings differed [the six time-points for seedlings after leaf rust inoculation were 0 h (Z11), 24 h (Z12), 48 h (Z12), 72 h (Z12), 96 h (Z12), and 168 h (Z13), but after mock inoculation only 0 h (Z11) and 168 h (Z13) were used]; mock-inoculated adult plants were treated as controls for adult plant resistance reaction. Inoculated seedlings and adult plants were also examined regularly on a daily basis during the above and some other time-points; phenotypic symptoms for susceptibility/ resistance were recorded (Supplementary Figure 1). Isolation of plant RNA, cDNA synthesis, and cDNA-AFLP analysis The methods used for isolation of RNA and cDNA synthesis were described earlier (Dhariwal et al. 2011). cDNA-AFLP
235
analysis was performed following Vos et al. (1995) and Bachem et al. (1996) with some modifications. A total of 16 EcoRI+3/MseI+3 primer combinations (each primer with three selective nucleotides) were used for cDNA-AFLP analysis (Supplementary Table 1). The details of the methods used for cDNA digestion, adaptor ligation, and PCR for cDNA-AFLP was described earlier (Dhariwal et al. 2011). Pictures of autoradiograms were taken using high definition camera (Canon, USA) and intensity of bands was analyzed using myImageAnalysis Software (Thermo Scientific, USA). DNA bands with presence/absence pattern or ≥2-fold change in intensity relative to control were considered as differentially expressed. Differentially expressed bands were excised from dried gels followed by DNA elution of transcript-derived fragments (TDFs) using standard protocols. The eluted TDFs were re-amplified using specific primers that were used for selective amplification during cDNA-AFLP, and were designated as a series of T. aestivum adult plant resistance gene Meerut (TaAPRGM). Cloning of TDFs, sequencing, and data processing The differentially expressed TDFs were cloned into pGEM-T easy vector (Promega Co., USA) directly or in pUC19 vector (Invitrogen, USA) following end-repairing using End-It™ DNA End-Repair Kit (EPICENTRE® Biotechnologies, Wi s c o n s i n) a n d t r an s f o r m e d in t o E . co li s t r ai n ElectroMAX™ DH 5α™ or DH 10B™ (Invitrogen, USA). The details of the methods used for cloning of TDFs were described earlier (Dhariwal et al. 2011). For each cloned TDF, five independent recombinant plasmids were sequenced using SP6/M13 primer on ABI Prism® 3700/3730xl Genetic Analyzer (Life Technologies Co, USA). All TDF sequences were deposited in the dbEST (GenBank accession numbers: JG392892 to JG392939). Isolation of genomic DNA from wheat stock CSP44 and from the pathogen Genomic DNA (gDNA) from leaves of adult plants of wheat stock CSP44 was isolated using standard CTAB method. Similarly, gDNA from the urediniospores of leaf rust fungus race 77-5 was isolated according to the protocol described by Wang et al. (2008). Functional analysis of TDFs Putative functions were assigned to TDFs by searching Gene Ontology (GO) and Enzyme Commission (EC) terms, and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations; Blast2GO software with blast, mapping, and annotation functions was used for this purpose (Conesa et al. 2005). GO terms for biological processes, molecular functions, and
236
cellular components were obtained using combined graph function of the software with default parameters. The KEGG analysis was performed using the KEGG annotation function and downloading the KEGG pathways. A keyword search with GO terms was performed against PathoPlant database. BLAST search of the TDF sequences was also performed against T. aestivum gene index/model (available in DFCI gene index, EnsemblPlants), TIGR TA assemblies, and P. triticina database to identify their similarity with genomic sequences of host (wheat) and the pathogen (leaf rust). TDFs with E value less than 1E-05 were treated as having significant similarity. Standard PCR analysis was carried out using primers designed from sequenced TDFs. The gDNA isolated from the host and the pathogen was used as template (Primer Express 3.0 was used for designing the primers; Supplementary Table 2); this facilitated confirmation of the source of individual TDFs (host vs. pathogen). The PCR products along with a 30-bp ultra low range DNA ladder were resolved on 2 % agarose gel (Fermentas, USA). Quantitative PCR analysis The quantitative PCR (qPCR) analysis was carried out with three technical replicates of RNA (from each of the two biological replicate samples) involving primers designed as above. ABI Prism® 7700 real time PCR platform from Life Technologies Co, USA was used for qPCR. Normalization of the amount of transcript accumulated for each TDF was carried out using wheat ‘glyceraldehyde-3-phosphate dehydrogenase’ gene (GPDH) as an internal control, and data were analyzed using 2-DDCt method (Higuchi et al. 1993; Livak and Schmittgen 2001). Statistical analysis Two tailed paired Student’s t test was used to test the significance of differential expression. The following comparisons were made for differential expression: (i) leaf rust-inoculated seedlings vs. leaf rust-inoculated adult plants, (ii) mockinoculated adult plants vs. leaf rust-inoculated adult plants, and (iii) mock-inoculated seedlings vs. leaf rust-inoculated seedlings. In silico chromosomal-arm assignment of differentially expressed wheat TDFs Nucleotide sequences of all differentially expressed wheat TDFs were BLAST searched against next generation survey sequences of wheat that were available chromosome arm-wise in IWGSC/URGI/Ensembl Plants database (International Wheat Genome Sequencing Consortium 2014). Sequences showing >98 % similarity and E value in the range of 0.0 to 1E-10 were considered having significant match, thus
Funct Integr Genomics (2015) 15:233–245
permitting assignment of TDFs to the corresponding arms of individual wheat chromosomes.
Results Disease development and host resistance responses After inoculation with the pathogen, the seedlings as well as the flag leaves of CSP44 were examined for the appearance of the disease, if any. It was found that only 120 h after inoculation (phase III), the seedlings exhibited pale-halo islands at the spore germinating sites (phase III). At 168 h, these pale-halo islands transformed into chlorotic spots that were associated with the emergence of pustules in the center of the chlorotic spots (Supplementary Figure 1). This eventually caused rupture of the epidermis along the surface of the inoculated leaf. However, at adult plant (booting) stage, no change on the leaf surface of flag leaves was observed till 96 h (phases I and II) following inoculation, and flaking started appearing at 120 h only (phase III) followed by full hypersensitive response (HR) at later stages (168 h onwards; Supplementary Figure 1). Identification of differentially expressed TDFs The cDNA-AFLP analysis of RNA samples collected from mock/pathogen-inoculated adult plants along the time-course of infection (0–168 h) led to the detection of 493 TDFs. The number of TDFs per primer combination ranged from 25 to 55 with an average of 31 (for a representative AFLP pattern, see Fig. 1). As many as 298 (60.44 %) of 493 TDFs exhibited differential expression [262 upregulated and 36 downregulated; relative to control (0 h after inoculation)] due to leaf rust inoculation in adult plants; the remaining 195 (39.56 %) of the 493 TDFs either showed non-specific expression variation due to both leaf rust inoculation and mock inoculation (107) or an indistinct expression (88) in adult plants. Functional annotation and identification of the source (host vs. pathogen) of differentially expressed TDFs In order to get insight into the functions and the source of differentially expressed TDFs due to leaf rust infection, 48 TDFs (40 upregulated and 8 downregulated) exhibiting high level of differential expression were cloned and sequenced. The sequences were used for GO annotation, and functions could be assigned to 42 (87.50 %) of these 48 TDFs; these 42 TDFs were classified into the following functional classes based upon MIPS Functional Catalogue: energy production, metabolism, transport, signaling, defense response, plant-pathogen interaction, transcriptional regulation, translation, and proteolysis (http://mips.
Funct Integr Genomics (2015) 15:233–245
Fig. 1 A representative autoradiogram of cDNA-AFLP analysis of 12 cDNA samples including six lanes (1, 3, 5, 7, 9, and 11) belonging to leaf rust-inoculated (I), and another six lanes (2, 4, 6, 8, 10, and 12) belonging to mock-inoculated (M) adult plants of wheat genotype CSP44 (six samples represents six time-points in each case). The differentially expressed bands of leaf rust-inoculated and mock-inoculated plants are shown by A and B respectively and that observed in both is shown by C
helmholtz-muenchen.de/funcatDB). Annotated GO terms were also used for keyword search against PathoPlant database but no significant matches were found. Details of the results of complete GO analysis including biological processes, molecular functions, cellular components, and KEGG pathways are given in Table 1, Supplementary Table 3, and Supplementary Figs. 2 and 3. The remaining six TDFs did not show GOBLAST match, GOMapping, and GOAnnotation during GO analysis. Thus, these transcripts apparently represent new candidate genes that were expressed following leaf rust infection. The sequences of all the 48 cloned TDFs were also used for BLAST search against the Puccinia Group Database. Sequences of four TDFs (TaAPRGM29, -31, -36, -37) exhibited similarity to four different supercontigs of P. triticina race BBBD1 (Table 1 and Supplementary Table 3) at the Puccinia Group Database. The PCR primers designed from these four TDF sequences showed amplification with the template gDNA of both wheat and P. triticina race 77–5 indicating the presence of conserved genomic regions in the host and the pathogen. qPCR for expression analysis of the response of host candidate genes during phase I to III In order to identify candidate genes involved in expression of resistance at adult plant stage, 18 TDFs showing
237
differential expression during cDNA-AFLP analysis were selected for qPCR analysis. These TDFs represented genes with either putative function or unknown functions mentioned earlier. The expression of these TDFs was quantified at both seedling (susceptible reaction) and adult plant (resistance reaction) stages. For 16/18 TDFs, differential expression was noticed in inoculated adult plants/seedlings during phases I and III relative to control (0 h) (Fig. 2a–r). The TDFs with high expression could be broadly grouped into three classes: (i) TDFs showing high expression in one treatment only: either during susceptible reaction in seedlings (TaAPRGM37; Fig. 2o), or during resistance reaction in adult plants (TaAPRGM1, -6, -22, -23, -25, -42; Fig. 2a, e, j, k, l, q) or in the mock-inoculated adult plants (TaAPRGM39; Fig. 2p); (ii) TDFs showing high expression in two or more treatments, but with a conspicuous high expression in a particular treatment [susceptible reaction in seedlings (TaAPRGM19; Fig. 2h), resistance reaction in adult plants (TaAPRGM3, -5, -8, -10, -26; Fig. 2c, d, f, g, m) or susceptible reaction in seedlings and mockinoculated adult plants (TaAPRGM36, -48; Fig. 2n, r)]; and (iii) TDFs showing an indistinct/stage-specific expression in all the treatments (TaAPRGM2, -21; Fig. 2b, i). It is apparent from the above results that although many TDFs were induced at adult plant stage, the expression of the following four TDFs was particularly high during susceptible reaction in the seedlings but was attenuated or remained near normal in adult plants (irrespective of the time-points used): TaAPRGM19, -36, -37, and -48. The expression was also examined at different timepoints. The following nine TDFs were induced during phase I, within 24 h after inoculation: TaAPRGM2, -3, 5, -6, -8, -21, -23, -26, -42 (Fig. 2b, c, d, e, f, i, k, m, q). Six of these nine TDFs (TaAPRGM3, -5, -6, -8, -23, -26), along with two others (TaAPRGM1, -19) were characterized by a constant change in their expression up to a specific time point (72 h/96 h/168 h; Fig. 2a, c, d, e, f, h, k, m). The expression of six TDFs, (TaAPRGM3, -5, -10, -22, -25, -26), including two of the above nine, returned to near basal level in phase III, during 96 to 168 h (Fig. 2c, d, g, j, l, m) after a dynamic or conspicuous high expression at 72 or 96 h; majority of the above TDFs had high expression at 96 h. Expression of TDFs at different time-points was also examined in the context of their function. For instance, TaAPRGM21, which is presumably involved in signaling, had high expression in phases I and III (i.e., 24 h; Fig. 2i), and TaAPRGM10 similarly involved in transcription regulation had high expression in phase II (i.e., 72 h; Fig. 2g). However, half of the TDFs including TaAPRGM8 that are presumably involved in energy release exhibited gradual increase in abundance up to phase III (i.e., 168 h; Fig. 2f).
Domain
JG392926 Phytepsin precursor-like (Aspartic proteinase) (H. vulgare) JG392929 Cytochrome P450 monooxygenase CYP72A16 (Z. mays)
TaAPRGM39
JG392896 TonB family protein
JG392912 Multidrug resistanceassociated protein MRP1 (T. aestivum)
TaAPRGM5
TaAPRGM22
3. Transport
TaAPRGM42
JG392897 Sucrose non-fermenting 4like protein (H. vulgare)
TaAPRGM6
TaAPRGM3
TaAPRGM1
2. Metabolism
JG392892 Predicted: inactive poly [ADP-ribose] polymerase RCD1-like (Brachypodium distachyon) JG392894 4-hydroxybenzoate nonaprenyltransferase
1.00e-44 –
1.60e-06 TonB_C
Electrochemical potentialdriven transport ATP catabolic process
Type-V protein secretion system complex Integral component of membrane
Oxidation-reduction process, Cytoplasmic membrane- Iron ion binding; electron carrier; oxidoreductase bounded vesicle; generation of precursor activity endoplasmic reticulum metabolites and energy Electrochemical potentialdriven transport activity ATP binding; ATPase activity; coupled to transmembrane movement of substances
Aspartic-type endopeptidase activity
4.10e-38 CypX
1.20e-06 –
Protein kinase activity
Membrane; mitochondria Transferase membrane
NAD+ ADP-ribosyl transferase activity; glycolysis
Cellular protein modification Cytoplasm process; response to stress, external stimulus; cell communication; regulation of biological process; metabolic process Lipid metabolic process; Vacuole proteolysis
Isoprene biosynthesis; ubiquinone biosynthesis; metabolic process
Nucleus
Mitochondria
Tricarboxylic acid cycle
Development; signal transduction; stress response
Catalytic activity
–
Pyruvate metabolism (4.4.1.5); MAPK signaling pathway
Oxidative phosphorylation (1.6.5.3)
KEGG pathway (EC)
ABC transporters
–
Brassinosteroid biosynthesis (1.14.13.112)
Lipid metabolism
–
Ubiquinone and other terpenoidquinone biosynthesis (2.5.1.39); biosynthesis of secondary metabolites; metabolic pathway
MAPK cascade; base excision repair (glycolysis; 2.4.2.30)
Oxoglutarate dehydrogenase Citrate cycle (TCA cycle; 1.2.4.2); Tryptophan (succinyl-transferring) metabolism; biosynthesis of activity; thiamine secondary metabolites pyrophosphate binding
NADH dehydrogenase (ubiquinone) activity
Molecular function(s)
Mitochondria
Cellular component
Mitochondrial electron transport; NADH to ubiquinone Metabolic process
Biological process
Gene ontology
7.10e-35 CBS
1.15e-03 PT_UbiA
3.03e-16 RST
2.31e-46 Glo EDI JG392913 Glyoxalase/bleomycin BRP-like resistance protein/ dioxygenase family protein 6.00e-09 UBN2 JG392925 2-oxoglutarate dehydrogenase E1 component, mitochondrial (Aegilops tauschii)
TaAPRGM23
TaAPRGM37
JG392899 NADH dehydrogenase subunit 4 and 5
9.10e-07 ND4 and 5
E value
TaAPRGM8
Accession DFCI gene index/TIGR TA no. assemblies/UniProt entry
Details of gene ontology (GO) results of 18 TDFs cloned from adult plant leaf rust-resistant genotype CSP44 carrying gene Lr48 and used for qPCR analysis
1. Energy production
Ta gene ID
Table 1
238 Funct Integr Genomics (2015) 15:233–245
JG392915 Calcium-dependent protein kinase 2
JG392893 No match
JG392909 SKP1/ASK1-like protein (T. aestivum)
JG392901 Hepatocyte growth factorregulated tyrosine kinase substrate-like protein (O. sativa)
En dash means no information available
TaAPRGM2
9. Unknown
TaAPRGM19
8. Proteolysis
TaAPRGM10
7. Transcriptional regulation
TaAPRGM25
Domain
3.40e-07 –
E value
–
Biological process
Gene ontology
Integral component of membrane
Cellular component
–
–
1.90e-21 L7/L12 Cterminal
9.60e-06 rpoB
7.50e-05 DUF3558
2.00e-53 LRR
–
Ubiquitin-dependent protein catabolic process; translation
DNA-directed RNA polymerase activity
Modulation by symbiont of defense-related host CPK2 pathway
–
–
Intracellular
Cytoplasm
–
Hydrolysis of peptide bonds
–
Calcium-dependent protein kinase activity
Protein kinase CK2 complex
–
–
–
Plant-pathogen interaction (CDPK)
–
–
ATP binding; carbohydrate binding; protein serine/ threonine kinase activity –
–
ABC transporters
KEGG pathway (EC)
Hydrolase activity; nucleotide binding
–
Molecular function(s)
–
JG392911 Gtp-binding protein Intracellular 2.60e-14 Gtp Nucleobase-containing [predicted membrane compound metabolic GTPase involved in stress process; catabolic process response (signal transduction mechanisms)] – JG392924 Lectin receptor kinase 5.00e-61 Oxidored q2 Stress response; innate (T. aestivum) immunity; ATP synthesis coupled electron transport
JG392916 Multidrug resistanceassociated protein MRP1 (T. aestivum)
Accession DFCI gene index/TIGR TA no. assemblies/UniProt entry
JG392935 TaLr1 cDNA clone TaLr1104A04 5' (T. aestivum) 6. Plant-pathogen interaction
TaAPRGM48
5. Defense response
TaAPRGM36
TaAPRGM21
4. Signaling
TaAPRGM26
Ta gene ID
Table 1 (continued)
Funct Integr Genomics (2015) 15:233–245 239
240
Funct Integr Genomics (2015) 15:233–245
a 30
TaAPRGM2 6
p (S/A) = 0.065 p (A/AM) = 0.078 p (S/ SM) = 0.281
Fold change
Fold change
b
TaAPRGM1 40 20 10
2 0
0h
48h
72h
96h
168h
d
TaAPRGM3 10 8 6 4 2 0
0h
Fold change
p (S/ A) = 0.473 p (A/AM) = 0.102 p (S/SM) = 0.262
48h
72h
96h
f
p (S/ A) = 0.075 p (A/AM) = 0.078 p (S/SM) = 0.006
48h
72h
96h
h Fold change
p (S/ A) = 0.465 p (A/AM) = 0.240 p (S/SM) = 0.304
0h
72h
96h
10 8 6 4 2 0
5
48h
72h
96h
168h
24h
48h
72h
96h
168h
15
p (S/ A) = 0.226 p (A/AM) = 0.298 p (S/SM) = 0.252
10 5 0
S A AM S A AM S A AM S A AM S A AM S A SM AM
0
24h
TaAPRGM22 20
Fold change
10
168h
p (S/ A) = 0.682 p (A/AM) = 0.060 p (S/SM) = 0.450
0h
j
p (S/ A) = 0.014 p (A/AM) = 0.027 p (S/SM) = 0.122
96h
TaAPRGM19
168h
TaAPRGM21 15
Fold change
48h
72h
0h
24h
48h
72h
96h
168h
S A AM S A AM S A AM S A AM S A AM S A SM AM
i
24h
48h
p (S/ A) = 0.056 p (A/AM) = 0.069 p (S/SM) = 0.346
0h
168h
TaAPRGM10 25 20 15 10 5 0
24h
TaAPRGM8 10 8 6 4 2 0
S A AM S A AM S A AM S A AM S A AM S A SM AM
Fold change
g
24h
168h
S A AM S A AM S A AM S A AM S A AM S A SM AM
0h
96h
p (S/ A) = 0.484 p (A/AM) = 0.130 p (S/SM) = 0.165
0h
Fold change
100 80 60 40 20 0
72h
S A AM S A AM S A AM S A AM S A AM S A SM AM
168h
S A AM S A AM S A AM S A AM S A AM S A SM AM
Fold change
24h
TaAPRGM6
48h
S A AM S A AM S A AM S A AM S A AM S A SM AM
0h
e
24h
TaAPRGM5 10 8 6 4 2 0
S A AM S A AM S A AM S A AM S A AM S A SM AM
Fold change
c
24h
S A AM S A AM S A AM S A AM S A AM S A SM AM
S A AM S A AM S A AM S A AM S A AM S A SM AM
0
p (S/ A) = 0.151 p (A/AM) = 0.019 p (S/SM) = 0.262
4
0h
24h
48h
72h
96h
168h
Fig. 2 (a–r) Histograms showing relative abundance of 18 transcripts analyzed using qPCR in CSP44 seedlings and adult plants at different time-points following inoculation with leaf rust pathogen P. triticina race 77-5 and also with mock (M) inoculation [S seedlings after P. triticina
inoculation (gray color), A adult plants after P. triticina inoculation (black color), SM seedlings after mock inoculation (white color), AM adult plants after mock inoculation (white color)]
In silico chromosomal assignment of TDFs
first instance, we confirmed the characteristic features of the wheat genotype CSP44, which is known to be immune to most predominant races of leaf rust in India at adult plant stage, although it is susceptible at the seedling stage, a characteristic associated with APR genes (Park and McIntosh 1994). Results of cDNA-AFLP and qPCR analyses involving the genotype CSP44 partly uncovered the molecular basis underlying resistance at the adult stage (APR), and susceptibility at the seedling stage; 10 TDFs were also assigned to individual chromosomes/arms (Supplementary Table 4). A comparison of the level of abundance of different transcripts belonging to selected TDFs at seedling and adult plant stages also allowed us to infer the changes in transcriptional activity of specific genes during expression of APR. Based on these results, we tend to speculate that the seedlings of CSP44 fail to
As many as 10 TDFs could also be assigned to 12 loci on 9 individual wheat chromosomes/arms (Supplementary Table 4). Only TaAPRGM36 had more than one (3) loci, which were not homoeologous but all belonged to subgenome A.
Discussion The present study was conducted to understand the molecular mechanism of adult plant resistance (APR), using the gene Lr48 carried by the wheat genotype CSP44. Therefore, in the
Funct Integr Genomics (2015) 15:233–245
k
l
TaAPRGM23 15
p (S/ A) = 0.155 p (A/AM) = 0.163 p (S/SM) = 0.490
Fold change
20
Fold change
241
10 5
0h
72h
96h
168h
0h
n
p (S/ A) = 0.185 p (A/AM) = 0.117 p (S/SM) = 0.139
Fold change
15 10 5 0
0h
24h
48h
72h
96h
Fold change
S A AM S A AM S A AM S A AM S A AM S A SM AM 72h
96h
0h
24h
48h
72h
96h
168h
24h
48h
72h
96h
168h
5 0
0h
24h
48h
72h
96h
168h
TaAPRGM48 120 100 80 60 40 20 0
p (S/ A) = 0.583 p (A/AM) = 0.380 p (S/SM) = 0.229
S A AM S A AM S A AM S A AM S A AM S A SM AM
Fold change
p (S/ A) = 0.104 p (A/AM) = 0.105 p (S/SM) = 0.259
168h
10
-5
r
TaAPRGM42 350 300 250 200 150 100 50 0
96h
p (S/ A) = 0.842 p (A/AM) = 0.330 p (S/SM) = 0.259
15
168h
S A AM S A AM S A AM S A AM S A AM S A SM AM
Fold change
q
48h
72h
TaAPRGM39 20
24h
48h
p (S/ A) = 0.207 p (A/AM) = 0.168 p (S/SM) = 0.097
0h
p
p (S/ A) = 0.424 p (A/AM) = 0.467 p (S/SM) = 0.148
0h
120 100 80 60 40 20 0
168h
TaAPRGM37 120 100 80 60 40 20 0
24h
TaAPRGM36
S A AM S A AM S A AM S A AM S A AM S A SM AM
-5
Fold change
48h
S A AM S A AM S A AM S A AM S A AM S A SM AM
Fold change
20
o
24h
TaAPRGM26
S A AM S A AM S A AM S A AM S A AM S A SM AM
m
p (S/ A) = 0.405 p (A/AM) = 0.293 p (S/SM) = 0.229
S A AM S A AM S A AM S A AM S A AM S A SM AM
S A AM S A AM S A AM S A AM S A AM S A SM AM
0
TaAPRGM25 50 40 30 20 10 0
0h
24h
48h
72h
96h
168h
Fig. 2 (continued)
activate defense measures, while the adult plants exhibit APR due to transcriptional regulation of a number of genes. Similar results associated with APR have also been reported earlier (Century et al. 1999; for a review, see Develey-Rivière and Galiana 2007). It is also apparent that among the differentially expressed genes, the upregulated genes outnumbered those which were downregulated (36) several folds, suggesting that the defense mechanisms involve activation of many, and repression of relatively fewer, specific genes/pathways (Cardoson et al. 2014). Activity of most of these genes/pathways was apparently initiated during phase I (0 to 24 h after inoculation) and reached a peak during phase II (24 to 96 h after inoculation), suggesting that phase II is the most crucial stage for determining/maintaining the resistance response by host genotype. The functions of 42 TDFs (resolved through GO annotation analysis) and their further classification (e.g., energy production, metabolism, defense, plant-pathogen interactions, etc.) suggested that these functions were largely no different from those reported in some earlier studies involving barleypowdery mildew, wheat-stripe rust, and wheat-leaf rust pathosystems (Caldo et al. 2004; Bolton et al. 2008; Coram et al. 2008; Mallard et al. 2008; Wang et al. 2010). These TDFs
also had multiple and diverse cellular locations (as revealed by GO cellular component analysis for 31 of 48 cloned TDFs) with overrepresentation in the mitochondria, chloroplast, and membrane systems indicating a global impact of Lr48-induced genes on cellular homeostasis, as also reported in the case of Arabidopsis thaliana following challenge with strains of Pseudomonas syringae (Torres et al. 2003). Functions of a few TDFs could not be resolved during the present study, which can be attributed either to their short length, or to their origin from UTRs or to their novelty, the corresponding sequences being absent in the databases (GenBank-NCBI, EMBL-EBI, UniProt, CerealsDB, URGI/EnsemblPlants, etc.). Apparently, these represented some novel candidate genes expressed during Lr48-mediated APR. The qPCR analysis revealed that after leaf rust infection, abundance of at least 11 TDFs (TaAPRGM1, -3, -5, -6, -8, 10, -22, -23, -25, -26, -42) improved and that of 4 TDFs (TaAPRGM19, 36, -37, -48) were declined or remained near normal at adult plant stage. Only six of the above 11 induced TDFs (TaAPRGM1,-6, -22, -23, -25, -42) exhibited highly significant difference in expression at the adult plant stage, suggesting their role in Lr48-mediated APR. These six TDFs are apparently involved in the following cellular activities/
242
Funct Integr Genomics (2015) 15:233–245
Seedling Stage
Adult Plant Stage N
A ?
?
N ND
NBS
Cp ND
? ROS
Nucleus
Nucleus
?
E
CC
CC
ND
Puccinia tricina infecon hyphae
NBS
LRR
Interplay of signals
N
Cp
RCD
TFs
ND
Mt
Mt
M C
?
ABA
G
G
ROS Expression of transporters, NADH dehydrogenase, defense proteins, etc.
RGA2 like LRR
RGA2 like
Puccinia tricina infecon hyphae
N
A
M
M P
C
C P
ABA
?
LecRK
LecRK Cytoplasm
ROS
Puccinia tricina infecon hyphae
Cytoplasm
ROS
Puccinia tricina infecon hyphae
Fig. 3 Proposed model for the possible mechanism of Lr48-mediated APR following seedling susceptibility. Pathways inferred during the present study are shown by solid arrows while those based on earlier studies are indicated by dotted arrows; highlighted (yellow color/with solid arrows) structures indicate activated molecules (for details, see text). LecRK lectin receptor kinase, G membrane-anchored GTPase
proteins, M MAPK (mitogen activated protein kinase), C CDPK (calcium-dependent protein kinase), A anti-microbial pumps or transporter, N NADPH oxidase, ND NADH dehydrogenase, RCD inactive poly (ADP-ribose) polymerase RCD1-like, Cp chloroplast, Mt mitochondria, E effector molecules, TFs transcription factors, ROS reactive oxygen species, ABA abscisic acid
processes: (i) metabolism [TaAPRGM1=inactive poly (ADPribose) polymerase RCD1-like, TaAPRGM6=sucrose nonfermenting 4-like protein, and TaAPRGM42=cytochrome P450 monooxygenase], (ii) transport (TaAPRGM26=multi drug resistance associate proteins), (iii) energy production (TaAPRGM23 = glyoxilase/bleomycin-resistant protein/ dioxigensae family protein), and (iv) plant-pathogen interaction (TaAPRGM25=CDPK-2). The magnitude and timing of appearance of these six TDFs suggest a coordinated expression pattern of CDPK and MAPK signaling (both known to be involved in innate immunity; Meszaros et al. 2006; Doczi et al. 2007), membrane-based transporters, and resistance proteins during APR in phase II (at 96 h). In the present study, three transcripts (TaAPRGM1, -23, -25) belonging to the above two signaling pathways exhibited elevated expression in adult plants at 96 h after inoculation (Fig. 2a, k, l), suggesting that these two pathways (in addition to other pathways discussed below) may be involved in Lr48-mediated APR also (Fig. 3). Seven different TDFs belonging to different secretory pathways were also identified during the present study (Table 1 and Supplementary Table 3); qPCR results for three of these seven TDFs (TaAPRGM5, -22, -26) were also available (Table 1), suggesting gradual/abrupt increase in their expression up to 96 h after inoculation (Fig. 2d, m). Therefore, the
genes corresponding to these three TDFs might be involved in secretory pathways that are relevant for Lr48-mediated APR. An involvement of secretary and/or transport machinery in extracellular immune responses has also been suggested in an earlier study (Kwon et al. 2008). Taken together, our results suggest that for imparting APR, genes are expressed in the following order: signaling genes were expressed first (within 24 h; e.g., TaAPRGM21) followed by genes belonging to transcription regulation, etc. (within 72 h; e.g., TaAPRGM10) and lastly the genes involved in metabolism, defense, etc. (at 72, 96, and 168 h; e.g., TaAPRGM1, -6, -8, -22, etc.). Another significant observation of the present study was also the decline in expression of several Lr48-responsive genes at later stages (phase III, Fig. 2); these genes had a distinctive expression in phase I and/or phase II (as discussed above), suggesting a dynamic but transient change in the transcriptional activity of a number of responded genes in the host. These results suggest that the recruitment as well as activation of various biological processes was purely transient in plants carrying an APR gene and thereafter transcript levels returned to near basal levels. However, this high level of transcriptional reprogramming during phase II imposes an additional energy demand on host machinery (Bolton et al. 2008). This also suggests that Lr48-mediated APR is an
Funct Integr Genomics (2015) 15:233–245
energy-intensive process, and this additional energy demand is met by recruitment of various energy releasing processes in phase II, which slowed down in phase III; these processes include the following: oxidative phosphorylation, pyruvate metabolism, and TCA cycle (see Table 1 and Supplementary Table 3 for energy producing genes reprogrammed). Similar expression pattern was reported in earlier studies involving wheat-leaf rust and Arabidopsis-bacterial speck pathosystems (Torres et al. 2003; Bolton et al. 2008). Association of specific PTI-, ETI-, and ROS-related TDFs in seedling susceptibility or APR An apparent association of reactive oxygen species (ROS) production/regulation-related genes either with seedling susceptibility or Lr48-mediated APR (Fig. 3) was also noticed. This observation was important, because it is known that ROS, particularly hydrogen peroxide (H2O2), after being produced due to pathogen attack, acts as a secondary signal molecule. At low concentrations, it activate MAPK protein and ABA-mediated stomatal closure (Fukao and BaileySerres 2004; Laloi et al. 2004; Mittler et al. 2004) and at high concentrations, it orchestrates programmed cell death/HR (Dat et al. 2000). One such TDF, TaAPRGM36, had enhanced expression in pathogen-inoculated seedlings as well as in mock-inoculated adult plants, in contrast to its negligible expression in pathogen-inoculated adult plants during phase II. This TDF seems to be derived from a PTI gene (lectin receptor kinase or LecRK), which inhibits ABA-mediated ROS production (Desclos-Theveniau et al. 2012), suggesting possible role of its elevated expression in loss of PTI, leading to susceptibility in seedlings, and that of its downregulation in improved ETI, imparting resistance in the adult plants (Fig. 3). This conclusion receives support from several reports including the following: (i) A suggested association of LecRKs with tolerance to biotic stresses through regulation of PTI (Bouwmeester and Govers 2009; Singh and Zimmerli 2013), (ii) association of the expression of resistance in mutants lacking LecRK with accumulation of reactive oxygen species (ROS) and constitutive stomatal closure (Desclos-Theveniau et al. 2012), (iii) involvement of G-type lectin receptor in providing resistance against Magnaporthe grisea that causes blast disease in rice (Chen et al. 2006). Two other interesting TDFs identified during the present study include TaAPRGM8 (encodes NADH dehydrogenase, a mitochondrial electron transport system enzyme) and TaAPRGM28 (encodes NADPH quinone oxidoreductase, a chlororespiration enzyme), which were presumably derived from genes responsible for the production of ROS (Bhattacharjee 2012). However, the expression of only TaAPRGM8 (but not that of TaAPRGM28) was studied. It showed increased transcriptional activity, apparently resulting in NADH dehydrogenase enzymatic activity continuously up
243
to 168 h in adult plants but not in seedlings, indicating its role in APR through ROS-mediated signaling/HR (Fig. 3). Another TDF (TaAPRGM1), which belongs to an inactive ‘poly (ADP-ribose) polymerase RCD1-like’ gene, also had high expression in pathogen-inoculated adult plants but not in pathogen-inoculated seedlings. Inactive ‘poly (ADP-ribose) polymerase RCD1-like’ gene is known to regulate oxidative stress genes (as positive regulator) and several other stress responsive genes (by interacting with transcription factors), and participate in abscisic acid, ethylene, and jasmonate signaling pathways in Arabidopsis (Overmyer et al. 2000, 2005; Ahlfors et al. 2004; Fujibe et al. 2004; Jaspers et al. 2009; Teotia et al. 2010; Teotia and Lamb 2011). Expression pattern of this gene, and the association of other genes with Lr48-mediated APR expression, also suggests that different stress-signaling pathways were cross-talking with each other, which may contribute to disease resistance (Manickavelu et al. 2010). Other genes reprogrammed in Lr48-mediated APR A number of TDFs, other than the few major genes discussed above, were also found associated with APR expression. These include four previously identified wheat genes which correspond to the following TDFs: (i) TaAPRGM6 (sucrose non-fermenting 4-like kinase which mediates ABA-induced changes in gene expression in response to stresses, GomezCadenas et al. 1999); (ii) TaAPRGM22 (multidrug resistanceassociated protein MRP1, Theodoulou et al. 2003); (iii) TaAPRGM25 (calcium-dependent protein kinase 2, Li et al. 2008), and (iv) TaAPRGM42 (cytochrome P450 monooxygenase, Krattinger et al. 2009)]. A new gene corresponding to TaAPRGM23 (glyoxalase/bleomycin resistance protein)] from the present study also belong to this class of genes that may be associated with APR. Elevated expression of all of the above genes was observed in pathogen-inoculated adult plants, but neither in pathogen-inoculated seedlings, nor in mock-inoculated adult plants, signifying their role in APR expression. Some other differentially expressed TDFs represent common transcripts expressed in disease resistance; these include those involved in metabolism (e.g., TaAPRGM3=4hydroxybenzoate nonaprenyltransferase; TaAPRGM40=catalase etc.), transport (e.g., TaAPRGM5=TonB family protein), signaling (e.g., TaAPRGM21=GTP-binding protein), defense (e.g., TaAPRGM31 = disease resistance protein RGA2), transcription (e.g., TaAPRGM10=hepatocyte growth factor-regulated tyrosine kinase), translation (e.g., TaAPRGM11=peptide chain release factor-2), and proteolysis (TaAPRGM19=SKP1/ASK1-like). Based on the above discussion involving results of the present study and the earlier published results, we tried to infer a network of changes/activities, which might be associated with the onset of resistance response in a wheat plant
244
carrying a gene for APR, like Lr48 used in the present study (Fig. 3). We hypothsize that at the seedling stage, inhibition of ABA-mediated ROS production caused by high expression of LecRK like protein leads to prolonged stomatal opening, thus facilitating the pathogen to infect host plant easily, leading to failure of PTI. In contrast, at the adult plant stage, through an unknown mechanism, pathogen attack leads to activation of NADPH oxidase (plasma membrane-based) and NADH dehydrogenase (mitochondrial) resulting in the following activities: release of ROS, closure of stomata, activation of CDPK2/MAPK proteins, membrane-based transporters, defense protein, etc. This in turn leads to expression of APR. Since no information was available hitherto about the genes that are induced due to recessive APR gene Lr48, the results of the present study enhance our knowledge towards understanding the molecular basis of leaf rust APR in wheat in general, and that of the Lr48-mediated APR in wheat genotype CSP44 in particular.
Acknowledgments PKG was awarded INSA Honorary Scientist and NASI Senior Scientist Fellowships during the tenure of this study. Thanks are due to the Department of Biotechnology, Government of India (grant no. BT/PR6037/AGR/02/308/05), for providing financial support to carry out this study. The authors are grateful to the Head, Regional Station, Directorate of Wheat Research, Flowerdale, Shimla, for providing inoculum of the leaf rust pathogen.
References Ahlfors R, Lang S, Overmyer K, Jaspers P, Brosche M, Tauriainen A, Kollist H, Tuominen H, Belles-Boix E, Piippo M, Inze D, Palva ET, Kangasjarvi J (2004) Arabidopsis RADICAL-INDUCED CELL DEATH1 belongs to the WWE protein-protein interaction domain protein family and modulates abscisic acid, ethylene, and methyl jasmonate responses. Plant Cell 16:1925–1937 Bachem CWB, van der Hoeven RS, de Bruijn SM, Vreugdenhil D, Zabeau M, Visser RGF (1996) Visualisation of differential gene expression using a novel method of RNA fingerprinting based on AFLP: analysis of gene expression during potato tuber development. Plant J 9:745–753 Bansal UK, Hayden MJ, Venkata BP, Khanna R, Saini RG, Bariana HS (2008) Genetic mapping of adult plant leaf rust resistance genes Lr48 and Lr49 in common wheat. Theor Appl Genet 117:307–312 Bhattacharjee S (2012) The language of reactive oxygen species signaling in plants. J Bot Article ID 985298, 22 pages Bolton MD, Kolmer JA, Xu WW, Garvin DF (2008) Lr34-mediated leaf rust resistance in wheat: transcript profiling reveals a high energetic demand supported by transient recruitment of multiple metabolic pathways. Mol Plant Microbe In 21:1515–1527 Bouwmeester K, Govers F (2009) Arabidopsis L-type lectin receptor kinases: phylogeny, classification and expression profiles. J Exp Bot 60:4383–4396 Caldo RA, Nettleton D, Wise RP (2004) Interaction-dependent gene expression in Mla-specified response to barley powdery mildew. Plant Cell 16:2514–2528 Cardoson DC, Martinati JC, Giachetto PF, Vidal RO, Carazzolle MF, Padilha L, Guerreiro-Filho O, Maluf MP (2014) Large-scale
Funct Integr Genomics (2015) 15:233–245 analysis of differential gene expression in coffee genotypes resistant and susceptible to leaf miner—toward the identification of candidate genes for marker assisted-selection. BMC Genomics 15:66 Century KS, Lagman RA, Adkisson M, Morlan J, Tobias R, Schwartz K, Smith A, Love J, Ronald PC, Whalen MC (1999) Developmental control of Xa21-mediated disease resistance in rice. Plant J 20:231–236 Chen X, Shang J, Chen D, Lei C, Zhou Y, Zhai W, Liu G, Xu J, Ling Z, Cao G, Ma B, Wang Y, Zhao X, Li S, Zhu L (2006) A B-lectin receptor kinase gene conferring rice blast resistance. Plant J 46:794–804 Cloutier S, McCallum BD, Loutre C, Banks TW, Wicker T, Feuillet C, Keller B, Jordan MC (2007) Leaf rust resistance gene Lr1, isolated from bread wheat (Triticum aestivum L.) is a member of the large psr567 gene family. Plant Mol Biol 65:93–106 Conesa A, Götz S, García-Gómez JM, Terol J, Talín M, Robles M (2005) Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21:3674–3676 Coram TE, Wang M, Chen X (2008) Transcriptome analysis of the wheat-Puccinia striiformis f. sp. tritici interaction. Mol Plant Pathol 9:157–169 Dat J, Vandenabeele S, Vranova E, Van Montagu M, Inze D, Van Breusegem F (2000) Dual action of the active oxygen species during plant stress responses. Cell Mol Life Sci 57:779–795 Desclos-Theveniau M, Arnaud D, Huang TY, Lin GJ-C, Chen W-Y, Lin Y-C, Zimmerli L (2012) The Arabidopsis lectin receptor kinase LecRK-V.5 represses stomatal immunity induced by Pseudomonas syringae pv. tomato DC3000. PLoS Pathog 8:e1002513 Develey-Rivière M-P, Galiana E (2007) Resistance to pathogens and host developmental stage: a multifaceted relationship within the plant kingdom. New Phytol 175:405–416 Dhariwal R, Vyas S, Govindraj BR, Jha SK, Khurana JP, Tyagi AK, Prabhu KV, Balyan HS, Gupta PK (2011) Analysis of differentially expressed genes in leaf rust infected bread wheat involving seedling resistance gene Lr28. Funct Plant Biol 38:479–492 Doczi R, Brader G, Pettko-Szandtner A, Rajh I, Djamei A, Pitzschke A, Teige M, Hirt H (2007) The Arabidopsis mitogen-activated protein kinase kinase MKK3 is upstream of group C mitogen-activated protein kinases and participates in pathogen signaling. Plant Cell 19:3266–3279 Dong X (1998) SA, JA, ethylene, and disease resistance in plants. Curr Opin Plant Biol 1:316–323 Feuillet C, Travella S, Stein N, Albar L, Nublat A, Keller B (2003) Mapbased isolation of the leaf rust disease resistance gene Lr10 from the hexaploid wheat (Triticum aestivum L.) genome. P Natl Acad Sci USA 100:15253–15258 Fofana B, Banks TW, McCallum B, Strelkov SE, Cloutier S (2007) Temporal gene expression profiling of the wheat leaf rust pathosystem using cDNA microarray reveals differences in compatible and incompatible defence pathways. Int J Plant Genomic, Article ID 17542: 13 pages Fujibe T, Saji H, Arakawa K, Yabe N, Takeuchi Y, Yamamoto KT (2004) A methyl viologen-resistant mutant of Arabidopsis, which is allelic to ozone-sensitive rcd1, is tolerant to supplemental ultraviolet-B irradiation. Plant Physiol 134:275–285 Fukao T, Bailey-Serres J (2004) Plant responses to hypoxia-is survival a balancing act. Trends Plant Sci 9:449–456 Giraud E, Ivanova A, Gordon CS, Whelan J, Considine MJ (2012) Sulphur dioxide evokes a large scale reprogramming of the grape berry transcriptome associated with oxidative signalling and biotic defence responses. Plant Cell Environ 35:405–417 Gomez-Cadenas A, Verhey SD, Holappa LD, Shen Q, Ho T-HD, WalkerSimmons MK (1999) An abscisic acid-induced protein kinase, PKABA1, mediates abscisic acid-suppressed gene expression in barley aleurone layers. Proc Natl Acad Sci USA 96:1767–1772 Higuchi R, Fockler C, Dollinger G, Watson R (1993) Kinetic PCR analysis: real time monitoring of DNA amplification reactions. Biotechnology 11:1026–1030
Funct Integr Genomics (2015) 15:233–245 Hua L (2009) Dissection of salicylic acid-mediated defense signaling networks. Plant Signal Behav 4:713–717 Huang L, Brooks SA, Li W, Fellers JP, Trick HN, Gill BS (2003) Mapbased cloning of leaf rust resistance gene Lr21 from the large and polyploid genome of bread wheat. Genetics 164:655–664 Hulbert SH, Bai J, Fellers JP, Pacheco MG, Bowden RL (2007) Gene expression patterns in near isogenic lines for wheat rust resistance gene Lr34/Yr18. Phytopathology 97:1083–1093 International Wheat Genome Sequencing Consortium (2014) A chromosome-based draft sequence of the hexaploid bread wheat genome. Science 345:1251788 Jaspers P, Blomster T, Brosche M, Salojaervi J, Ahlfors R, Vainonen JP, Reddy RA, Immink R, Angenent G, Turck F, Overmyer K, Kangasjaervi J (2009) Unequally redundant RCD1 and SRO1 mediate stress and developmental responses and interact with transcription factors. Plant J 60:268–279 Jones JDG, Dangl JL (2006) The plant immune system. Nature 444:323–329 Khanna R, Bansal UK, Saini RG (2005) Genetics of adult plant stripe rust resistance in CSP44, a selection from Australian wheat. J Genet 84: 337–340 Krattinger SG, Lagudah ES, Spielmeyer W, Singh RP, Huerta-Espino J, McFadden H, Bossolini E, Selter LL, Keller B (2009) A putative ABC transporter confers durable resistance to multiple fungal pathogens in wheat. Science 323:1360–1363 Kumar S, Wang Z, Banks TW, Jordan MC, McCallum BD, Cloutier S (2014) Lr1-mediated leaf rust resistance pathways of transgenic wheat lines revealed by a gene expression study using the Affymetrix GeneChip® Wheat Genome Array. Mol Bree. doi:10.1007/s11032-014-0022-6 Kunkel BN, Brooks DM (2002) Cross talk between signaling pathways in plant defense. Curr Opin Plant Biol 5:325–331 Kwon C, Bednarek P, Schulze-Lefert P (2008) Secretory pathways in plant immune responses. Plant Physiol 147:1575–1583 Laloi C, Apel K, Danon A (2004) Reactive oxygen signaling: the latest news. Curr Opin Plant Biol 7:323–328 Li AL, Zhu YF, Tan XM, Wang X, Wei B, Guo HZ, Zhang ZL, Chen XB, Zhao GY, Kong XY, Jia JZ, Mao L (2008) Evolutionary and functional study of the CDPK gene family in wheat (Triticum aestivum L.). Plant Mol Biol 66:429–443 Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2–DDCT method. Methods 25:402–408 Mallard S, Nègre S, Pouya S, Gaudet D, Lu ZX, Dedryver F (2008) Adult plant resistance-related gene expression in ‘Camp Remy’ wheat inoculated with Puccinia striiformis. Mol Plant Pathol 9:213–225 Manickavelu A, Kawaura K, Oishi K, Shin-I T, Kohara Y, Yahiaoui N, Keller B, Suzuki A, Yano K, Ogihara Y (2010) Comparative gene expressin analysis of susceptible and resistant near-isogenic lines in common wheat infected by Puccinia triticina. DNA Res 17:211–222 Md T, Sanchez P, Fernandez-Delmond I, Grant M (2003) Expression profiling of the host response to bacterial infection: the transition from basal to induced defence responses in RPM1-mediated resistance. Plant J 33:665–676 Meszaros T, Helfer A, Hatzimasoura E, Magyar Z, Serazetdinova L, Rios G et al (2006) The Arabidopsis MAP kinase kinase MKK1 participates in defence responses to the bacterial elicitor flagellin. Plant J 48:485–498 Mittler R, Vanderauwera S, Gollery M, Van Breusegem F (2004) Reactive oxygen gene network of plant. Trends Plant Sci 9:490–498 Mur LAJ, Elena P, Sandra P, Anthony HM, Henrik HK (2013) Integrating nitric oxide into salicylic acid and jasmonic acid/ethylene plant defence pathways. Front Plant Sci 4:1–7 Nandi A, Kachroo P, Fukushige H, Hildebrand DF, Klessig DF, Shah J (2003) Ethylene and jasmonic acid signaling affect the NPR1independent expression of defense genes without impacting resistance to Pseudomonas syringae and Peronospora parasitica in the Arabidopsis ssi1 mutant. Mol Plant Microbe In 16:588–599
245 Overmyer K, Tuominen H, Kettunen R, Betz C, Langebartels C, Sandermann H Jr, Kangasjaervi J (2000) Ozone-sensitive arabidopsis rcd1 mutant reveals opposite roles for ethylene and jasmonate signaling pathways in regulating superoxide-dependent cell death. Plant Cell 12:1849–1862 Overmyer K, Brosche M, Pellinen R, Kuittinen T, Tuominen H, Ahlfors R, Keinaenen M, Saarma M, Scheel D, Kangasjaervi J (2005) Ozone-induced programmed cell death in the Arabidopsis radicalinduced cell death1 mutant. Plant Physiol 137:1092–1104 Park RF, McIntosh RA (1994) Adult plant resistances to Puccinia recondita f.sp. tritici in wheat. New Zeal J Crop Hort 22:151–158 Park RF, Mohler V, Nazari K, Singh D (2014) Characterisation and mapping of gene Lr73 conferring seedling resistance to Puccinia triticina in common wheat. Theor Appl Genet 127:2041–2049 Saari EE, Prescott JM (1985) World distribution in relation to economic losses. In: Roelfs AP, Bushnell WR (eds) The cereal rusts, vol. ii: diseases, distribution, epidemiology, and control. Academic, Orlando, pp 259–298 Saini RG, Kaur M, Singh B, Sharma S, Nanda GS, Nayar SK, Gupta AK, Nagarajan S (2002) Lr48 and Lr49, noval hypersensitive adult plant leaf rust resistance genes in wheat (Triticum aestivum L.). Euphytica 124:365–370 Singh P, Zimmerli L (2013) Lectin receptor kinases in plant innate immunity. Front Plant Sci 4:1–4 Singh A, Pallavi JK, Gupta P, Prabhu KV (2011) Identification of microsatellite markers linked to leaf rust adult plant resistance (APR) gene Lr48 in wheat. Plant Breed 130:31–34 Singh D, Mohler V, Park RF (2013) Discovery, characterisation and mapping of wheat leaf rust resistance gene Lr71. Euphytica 190:131–136 Spielmeyer W, Mago R, Wellings C, Ayliffe M (2013) Lr67 and Lr34 rust resistance genes have much in common—they confer broad spectrum resistance to multiple pathogens in wheat. BMC Plant Biol 13: 96 Teotia S, Lamb RS (2011) RCD1 and SRO1 are necessary to maintain meristematic fate in Arabidopsis thaliana. J Exp Bot 62:1271–1284 Teotia S, Muthuswamy S, Lamb RS (2010) RADICAL-INDUCED CELL DEATH1 and SIMILAR TO RCD ONE1 and the stress-induced morphogenetic response. Plant Signal Behav 5:143–145 Theodoulou FL, Clark IM, He X-L, Pallett KE, Cole DJ, Hallahan DL (2003) Xenobiotics induce glutathione transferases and multidrug resistance associated protein in wheat. Pest Manag Sci 59:202–214 Thomma BP, Penninckx IA, Broekaert WF, Cammue BP (2001) The complexity of disease signaling in Arabidopsis. Curr Opin Immunol 13:63–68 Van der Does D, Leon-Reyes A, Koornneef A, Van Verk MC, Rodenburg N, Pauwels L, Goossens A, Körbes AP, Memelink J, Ritsema T, Van Wees SCM, Pieterse CMJ (2013) Salicylic acid suppresses jasmonic acid signaling downstream of SCFCOI1-JAZ by targeting GCC promoter motifs via transcription factor ORA59. Plant Cell 25:744–761 Vos P, Hogers R, Bleeker M, Reijans M, de Lee TV, Hornes M, Frijters A, Pot J, Peleman J, Kuiper M, Zabeau M (1995) AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res 23:4407– 4414 Wang XJ, Zheng WM, Buchenauer H, Zhao J, Han QH, Huang LL, Kang ZS (2008) The development of a PCR-based method for detecting Puccinia striiformis latent infections in wheat leaves. Eur J Plant Pathol 120:241–247 Wang X, Liu W, Chen X, Tang C, Dong Y, Ma J, Huang X, Wei G, Han Q, Huang L, Kang Z (2010) Differential gene expression in incompatible interaction between wheat and stripe rust fungus revealed by cDNAAFLP and comparison to compatible interaction. BMC Plant Biol 10:9 Zadok JC, Chang TT, Konzak FC (1974) A decimal code for growth stages of cereals. Weed Res 14:415–421 Zhang L, Meakin H, Dickinson M (2003) Isolation of genes expressed during compatible interactions between leaf rust (Puccinia triticina) and wheat using cDNA-AFLP. Mol Plant Pathol 4:469–477
