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TaULP5 contributes to the compatible interaction of adult plant resistance wheat seedlings-stripe rust pathogen
Physiological and Molecular Plant Pathology 96 (2016) 29e35
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Physiological and Molecular Plant Pathology journal homepage: www.elsevier.com/locate/pmpp
TaULP5 contributes to the compatible interaction of adult plant resistance wheat seedlings-stripe rust pathogen Hao Feng a, Qiuling Wang b, Xiaoqiong Zhao b, Lina Han b, Xiaojie Wang a, Zhensheng Kang a, * a b
College of Plant Protection, State Key Laboratory of Crop Stress Biology for Arid Areas, Northwest A&F University, Yangling, Shaanxi, 712100, China College of Life Sciences, Northwest A&F University, Yangling, Shaanxi, 712100, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 March 2016 Received in revised form 16 June 2016 Accepted 27 June 2016 Available online 29 June 2016
Adult plant resistance indicates that plant is susceptible to pathogen at seedling stage, but resistant at adult stage. Understanding the mechanism of the interactions between APR wheat plants and Puccinia striiformis f. sp. tritici (Pst) is important for the creation of strategies to improve cultivar disease resistance. In this study, a full-length cDNA was isolated from APR wheat cultivar Xingzi 9104 (XZ), and was designated as ubiquitin-like protein 5 (TaULP5). TaULP5 was likely to be located in the cytoplasm, with a percentage of 75.9% Arabidopsis protoplasts number. The expression of TaULP5 was largely induced in the compatible interaction of wheat seedlings to Pst, while no obvious change was found in the incompatible interaction of wheat adult plants to Pst. Moreover, when TaULP5 was knocked down, the wheat resistance at seedling stage to Pst was improved. In addition, knockdown of TaULP5 increased the expression levels of some biotic stress-related genes, such as PR1 and PR2. It is the first time to confirm that ubiquitin-like protein could contribute to the compatible interaction of XZ to Pst, and the results will lay a foundation for understanding the mechanisms of different interactions between APR wheat plants and Pst at posttranslational level. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Abiotic stress Adult plant resistance Puccinia striiformis f. sp. tritici Ubiquitin-like protein 5 Wheat
1. Introduction Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most destructive wheat diseases in the world. Breeding and rational utilization of disease-resistant varieties is the safest, the most effective, the most economical and the most environmentally sound method for controlling wheat stripe rust [5,8]. At present, wheat resistance to Pst can be broadly categorized into allstage resistance (seedling resistance) and adult plant resistance (APR). Compared with seedling resistance, APR is much more stable and durable. APR indicates that plant is susceptible to pathogen at seedling stage, but resistant at adult stage. Understanding the
Abbreviations: dpvi, days post virus inoculation; EF, elongation factor 1 alphasubunit gene; EST, expressed sequence tag; hpi, hours post-inoculation; ORFs, Open reading frames; PcG, Polycomb group; PDS, phytoene desaturase gene; Pst, Puccinia striiformis f. sp. tritici; RT-PCR, Reverse-transcription polymerase chain reaction; UBLs, Ubiquitin-like proteins; UBP, ubiquitin-specific protease gene; ULP5, Ubiquitin-like protein 5; UPS, ubiquitin-proteasome system; VIGS, virus-induced gene silencing; XZ, Xingzi9104. * Corresponding author. E-mail address: [email protected] (Z. Kang). http://dx.doi.org/10.1016/j.pmpp.2016.06.008 0885-5765/© 2016 Elsevier Ltd. All rights reserved.
mechanisms of different interactions between APR wheat and Pst is important for the strategies creation of improving cultivar disease resistance. Post-translational modification of proteins through the ubiquitin-proteasome system (UPS) serves a critical regulatory role in most cellular processes [7,9,20,22]. Ubiquitins, as a group of highly conserved eukaryotic polypeptides 76 amino acids in length, play roles by attaching to lysine residues (or sometimes to other amino acids) of their target proteins via their C-terminal glycines [12,15]. Ubiquitin-like proteins (UBLs) are another important component of the UPS. And there is a resemblance between UBLs and ubiquitins in sequence [16]. All UBLs share a similar threedimensional structure [25]. UBLs are ligated to their target proteins or other molecules by distinct but evolutionarily related enzyme cascades [18]. Host UBL pathways have been confirmed to play important roles in plant-pathogen interactions. For example, the ubiquitin-like protein ISG15 was found to be involved in the host antiviral immune defense [24,31]. Ubiquitin-like protein 5 (ULP5) is a recently identified component of the UPS. UBL5 was first identified in 2001 as an 8.5-kD protein product of a gene isolated from human adult iris cDNAs
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[13]. Unlike other ubiquitins, ULP5 contains an ubiquitin super-fold with an electrostatic surface [23]. Meanwhile, the C-terminal peptide sequence of ULP5 is not a double glycine but a pair of tyrosines, followed with variable amino acid residues [29]. According to previous report, ULP5 may affect rat energy metabolism [2]. In addition, the ULP5 yeast ortholog ubiquitin-1 (Hub1) had been reported in Saccharomyces cerevisiae, and it was found to play a role in mRNA and pre-mRNA splicing [29]. However, the studies about the function of plant ULP5 were relatively backward. Little is known regarding the biological functions of plant ULP5, although many ULP5 genes have been predicted based on the ESTs and genomic information (NCBI) of some plants. Thus, whether ULP5 can participate in plant disease resistance, especially the interactions of APR plant and pathogen? In this study, TaULP5 was firstly isolated from APR cultivar Xingzi 9104 (XZ), and the gene function in the compatible interaction between XZ and Pst was explored. The results will lay a foundation for understanding the mechanisms of different interactions between APR wheat plants and Pst at post-translational level. 2. Materials and methods 2.1. Plant materials and treatments XZ processes APR to Pst pathotype CYR32, that means it is susceptible to CYR32 at the seedling stage but resistant at the adult stage [19]. XZ and CYR32 used in this study were obtained from the Institute of Plant Pathology, Northwest A&F University, Yangling, Shaanxi, China. Wheat cultivation and Pst inoculation were performed as described in Ref. [10]. Leaves were collected at 0, 24, 48 and 120 h post-inoculation (hpi). After collection, all samples were immediately frozen in liquid nitrogen and stored at 80 C. 2.2. RNA extraction and cDNA synthesis Extraction of total RNA was performed with TRIzol reagent (Invitrogen, Carlsbad, CA) following the manufacturer's instructions. DNase I was used to remove the genomic DNA from total RNA. The total RNA was quantitated using a NanoDrop™ 1000 spectrophotometer (Thermo Fisher Scientific, USA). Three mg of total RNA was used to synthesize the first-strand cDNA using the RT-PCR system (Promega, Madison, WI, USA) with the Oligo (dT) 18 primer according to the manufacturer's instructions. 2.3. Gene isolation and sequence characterization The sequence fragment identified from the transcriptome libraries of XZ challenged with Pst was used as a query probe to screen publically available wheat expressed sequence tag (EST) databases for the full length of target gene. Homologous wheat EST sequences were retrieved and assembled. Open reading frames (ORFs) in the assembled sequences were predicted by NCBI's ORF FINDER. Reverse-transcription polymerase chain reaction (RT-PCR) was used to amplify the full-length cDNA with specific primers (Table A.1). The PCR products were cloned into the pMD18-T Simple vector (TaKaRa Biotechnology) and sequenced with an ABI PRISM 3130xl Genetic Analyzer (Applied Biosystems, USA). The DNAMAN (version 5.2.2) program was used for phylogenetic comparison between the deduced protein sequences and the corresponding characterized proteins from other plant species. 2.4. Subcellular localization of TaULP5 The TaULP5 ORF was PCR-amplified without stop codons using specific primers with HindIII and BamHI restriction sites. The
sequence fragment was then sub-cloned into PTF486, which contains the eGFP open reading frame. The recombinant vector PTF486-TaULP5 was end-sequenced with an ABI PRISM 3130XL Genetic Analyzer (Applied Biosystems, USA). The primers used for vector construction were listed in Table A.1. Well-expanded leaves from 4 week-old Arabidopsis thaliana were used for protoplast preparation. Mesophyll protoplasts and reagents were prepared according to [30]. The PTF486-TaULP5-eGFP fusion construct was transformed into Arabidopsis thaliana mesophyll protoplasts using the method described by Ref. [30]. Protoplasts were incubated with plasmids at 25 C in the dark for 16 h with gentle swirling at 40 rpm. eGFP fluorescence images of transformed protoplasts were obtained using confocal microscopy with a Nikon PCM2000 (BioRad) laser-scanning confocal imaging system. The experiment was repeated every other day using the same batch of Arabidopsis plants for three times totally. 2.5. Quantitative RT-PCR (qRT-PCR) The expression profiles of TaULP5 and PR protein genes were determined using quantitative real-time PCR analyses with specific primers (Table A.1). These primers were first used to amplify the fragment by regular PCR. The cloned fragment was then sequenced to confirm primer specificity. The wheat translation elongation factor 1 alpha-subunit (EF) gene (GenBank Accession No. M90077) and cytoplasmatic ribosomal protein S13 gene (GenBank Accession No. AY736126) were selected as the internal reference genes for qRT-PCR analyses. Three independent biological replicates were used for each time point, as well as a non-template control, and the results were analyzed using the comparative threshold (2DDCT) method [21]. 2.6. Functional analysis of TaULP5 in response to Pst infection For virus-induced gene silencing (VIGS) vector construction, a 223-bp fragment was amplified using specific primers with PacI and NotI restriction sites. The TaPDS sequence fragment in BSMVTaPDS was replaced by the amplicon to generate the BSMV-TaULP5 vector. The recombinant vectors were end-sequenced with an ABI PRISM 3130XL Genetic Analyzer (Applied Biosystems, USA). The primers used for vector construction were listed in Table A.1. The detailed VIGS procedures were performed as described by Ref. [27] with a minor revision. In vitro transcription products of the three BMSV genome sequences (a, b, g) were diluted 30 times, and 0.5 mL of each was mixed in a 1:1:1 ratio. Next, 9 mL of FES buffer was added to the mixture. And then the mixtures were directly applied to the second leaves by rub inoculation with a gloved finger. BSMVTaPDS was used to silence the wheat phytoene desaturase gene (PDS) as a positive control and FES buffer was used as a negative control. The wheat seedlings inoculated with BSMV were incubated in a growth chamber at 25 ± 2 C. Nine days post inoculation (dpi), the base of the third leaves and the top of the fourth leaves were infected with CYR32. After Pst inoculation, the wheat seedlings were incubated in a growth chamber at 16 ± 2 C. The Pst-inoculated leaves were then sampled at 0, 24, 48, 120 hpi for silencing efficiency analysis and histological observation. Pst infection types were examined at 15 dpi. Two independent biological replicates were performed for each treatment. 2.7. Histological observation The samples were stained as previously described [28], and the stained leaf segments were observed to analyze the infection site, necrotic area, and hyphal length using an Olympus BX-51 microscope (Olympus Corp., Tokyo). No more than 5 different infection sites were examined on each randomly selected leaf segment, and a
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total of 45 infection sites were examined per treatment. Statistical analysis was performed with SPSS (Statistical Package for the Social Sciences) software. 3. Results 3.1. The identification of TaULP5 The sequence fragment identified from the transcriptome libraries of XZ challenged with Pst (data not publihsed) was used as a query to screen wheat EST database for full-length gene sequence.
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A 292-bp wheat cDNA sequence was identified in silico and amplified by RT-PCR; this sequence contains an open reading frame encoding a 73-amino acid polypeptide (Fig. 1a). Analysis of a multisequence alignment showed highly conservation among the deduced protein and its homologs from other plants. Moreover, the C-terminus of the deduced peptide sequence is a pair of tyrosines, which is the typical characteristic of ULPs (Fig. A.1). In addition, the phylogenetic analysis showed that the candidate gene was clustered with the ubiquitin-like protein 5 genes from Brachypodium sylvaticum and Hordeum vulgare (Fig. 1b). Therefore, we designated the candidate gene as wheat ubiquitin-like protein 5 (TaULP5).
Fig. 1. Isolation of TaULP5 and characterization of the deduced amino acid sequence. (a) The cDNA fragment of TaULP5 was identified by in silico cloning and sequencing. The longest ORF was 222 bp, encoding a protein of 73 amino acids. The underlined sequences indicate gene-specific primers for RT-PCR, and the arrows indicate the direction of amplification. The triple bases in the grey boxes are the start and stop codons. (b) A phylogenetic tree of TaULP5 constructed with the multiple alignment program DNAMAN. The branches are labeled with the gene names and GenBank accession numbers from different plant species. The GenBank accession numbers are as follows: wheat XZ, KJ476506; Arabidopsis thaliana, NP_190104.1; Brachypodium distachyon, XP_003578938.1; Cucumis sativus, XP_004139444.1; Glycine max, XP_003535365; Hordeum vulgare, ABO42268.1; Medicago truncatula, XP_003601666.1; Oryza sativa, NP_001176788.1; Theobroma cacao, XP_007016757.1; Vitis vinifera, XP_002280046.1; Zea mays, NP_001150362.1.
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3.2. TaULP5 is localized to the cytoplasm To verify the subcellular localization of TaULP5, the TaULP5 gene was fused to green fluorescent protein (eGFP). The fusion vector PTF486-TaULP5-eGFP and the control vector PTF486-eGFP were transformed into Arabidopsis thaliana mesophyll protoplasts. Fluorescence microscopic observation of transformed cells showed that the green fluorescent of 85.7% control protoplasts was distributed in the whole cell of control protoplasts, including the cytoplasm and nucleus; and the TaULP5-eGFP fusion protein was likely to be accumulated in the cytoplasm, with a percentage of 75.9% protoplasts number (Fig. 2). 3.3. TaULP5 is highly expressed in the compatible wheat-Pst interaction To investigate the function of TaULP5 in APR wheat response to
Pst, the transcript profile was evaluated by qRT-PCR in both XZ seedlings and adults challenged with Pst using two inner reference genes. TaULP5 was stably expressed with no significant changes from 24 to 120 hpi compared to the control (0 hpi) in the adult stage. However, expression of TaULP5 was significantly upregulated in XZ seedlings response to Pst (Fig. 3). 3.4. Knocking down of TaULP5 improves the resistance of XZ seedlings to Pst To identify the function of TaULP5 in the compatible interaction between XZ seedlings and Pst, a barley stripe mosaic virus (BSMV)based VIGS system was used to silence the expression of TaULP5. All BMSV-inoculated plants displayed mild chlorotic mosaic symptoms at 7 days post virus inoculation (dpvi), and no obvious defect in leaf growth was observed (Fig. 4b). Typical photobleaching phenotypes occurred on BSMV-TaPDS-inoculated plants at 12 dpvi (Fig. 4c). The
Fig. 2. Detection and subcellular localization of the TaULP5-eGFP fusion protein in mesophyll protoplasts of Arabidopsis. (a) Vector indicates the positive control, and the GFP signal was detected throughout the cell, including the nucleus. ULP represents a fusion protein, which was located in the cytoplasm. W: image of the protein under white light; M: image of the fusion protein under BW fluorescence. The GFP signal is indicated in green, and chlorophyll autofluorescence is show in red; GFP: image of the fusion protein under GW light. The GFP signal is indicated in green. Bar ¼ 10 mm. (b) Quantitation of the nucleus-versus-cytoplasm partition of the TaULP5-eGFP fusion protein as shown in (a). ‘Cytoplasm’ indicates the GFP signal was mainly located in cytoplasm, ‘nucleus-cytoplasm’ indicates the GFP signal could be observed in both nucleus and cytoplasm obviously, and ‘other’ means no typical location signal was observed.
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Fig. 3. qRT-PCR expression profiles of TaULP5 in wheat leaves in response to Pst pathotype CYR32. qRT-PCR data were normalized to the expression level of the wheat elongation factor 1 alpha-subunit (EF) gene (a) and cytoplasmatic ribosomal protein S13 (b). The relative expression levels of TaULP5 at different growth stages of plants challenged with Pst were calculated using the comparative threshold (2DDCT) method. The mean values and standard deviations were calculated with data from three independent replicates.
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infected with BSMV-TaULP5. Although the phenotype of TaULP5silenced wheat plants was still considered to be susceptible, the resistance level was obviously increased (f). To confirm whether the phenotypic changes were caused by the silence of TaULP5, qRT-PCR was used to examine the relative transcript level of TaULP5. Compared with BMSV-g-inoculated wheat leaves, the expression of TaULP5 was reduced 89%, 84% and 85% at 24, 48 and 120 hpi, respectively (Fig. 5a). Moreover, the transcript levels of four PR genes (PR1, PR2, PR3 and PR5) were also detected in TaULP5-silenced plants after inoculated with Pst. The transcript levels of TaPR1 and TaPR2 were up-regulated in TaULP5-knockdown leaves at 24 hpi, for approximately 2.6 and 6.0 fold increased, respectively. At 48 hpi, these two PR genes showed similar expression levels with BMSV-g inoculated wheat leaves. However, the expression of TaPR3 and TaPR5 showed no significant change (Fig. 5b). To further explore the enhanced stripe rust fungus resistance of TaULP5-silenced wheat leaves, hyphal growth and cell death were examined at 24, 48 and 120 hpi microscopically. As shown in Fig. 6 and Table 1, hyphal length showed no significantly difference at 24 and 48 hpi compared with that of the mock-inoculated and BSMVg-infected plants. In contrast, hyphae length in TaULP5-silenced wheat leaves was significantly shorter (P < 0.05) at 120 hpi. Moreover, larger necrotic areas around the Pst infection sites were observed at 48 and 120 hpi in the TaULP5-silenced plants. It is likely that the suppression of TaULP5 may facilitate host cell death, and hyphal growth of stripe rust pathogen was suppressed by the resistant reaction of plant.
4. Discussion Plant diseases are a major factor restricting food production worldwide. Rational utilization of APR is much more stable and durable for disease control. Thus, exploring the molecular mechanisms of APR plant-pathogen interactions is necessary for the generation of new disease-control strategies. Previous studies have found that ubiquitin-proteasome system (UPS) plays a critical
Fig. 4. Phenotype of TaULP5-knockdown leaves from Pst-challenged wheat plant seedlings. For functional analysis of TaULP5, virus-induced gene silencing (VIGS) was applied to seedling-stage wheat plants. No change was found in the plant preinoculated with FES buffer (a). All of the BSMV-g-infected plants displayed mild chlorotic mosaic symptoms at 7 days post virus inoculation (dpvi) in the seedling stage (b). Photobleaching was observed at 12 dpvi when the leaves were inoculated with the positive control combination of BSMV-TaPDS vectors (c). A typical phenotype was observed when leaves pre-inoculated with FES buffer and BSMV-g were challenged with CYR32 (d and e). Phenotypic changes were observed in TaULP5-knockdown leaves (f) at 14 days post-inoculation of Pst.
leaves of the mock-inoculated plants and those infected with BSMV-g and BSMV-TaULP5 were further inoculated with CYR32. Fourteen days later, a fully compatible phenotype was observed on leaves of the plants mock inoculated with FES buffer and infected with BSMV-g (Fig. 4d and e). However, except for slight sporulation, typical necrotic spots emerged on leaves of the plants previously
Fig. 5. Silencing efficiency of TaULP5 and PR proteins expression in TaULP5knockdown leaves as measured by qRT-PCR. The silencing efficiency of TaULP5 (a) and the relative expression levels of PR proteins (b) in TaULP5-knockdown leaves challenged with pathotypes CYR32 were calculated using the comparative threshold (2DDCT) method. The data were normalized to the wheat elongation factor 1 alphasubunit (EF) expression level. Mean values and standard deviations were calculated with data from two independent replicates.
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Fig. 6. Histological observation of host cells and pathogen hyphal change in the TaULP5-knockdown leaves responding to Pst. The TaULP5-knockdown leaves and control leaves challenged with Pst pathotype CYR32 were sampled at 24, 48, and 120 hpi. The hyphae were stained to facilitate analysis of the length change between control leaves (A, B, C) and TaULP5-knockdown leaves (a, b, c). The histological changes were observed under an epifluorescence microscope. D-F and d-f indicate the change in necrotic cell area at 24, 48 and 120 hpi in the control and TaULP5-knockdown leaves, respectively. Bar ¼ 20 mm. HMC, haustorial mother cell; IH, initial hypha; NC, necrotic cells; SH, second hypha; SV, substomatal vesicle.
Table 1 Histological analysis of wheat TaULP5 knockdown leaves responding to Pst pathotype CYR32*. Treatmenta
BSMV-g BSMV-TaULP5
Necrotic areab
Hyphal lengthc
24 hpi
48 hpi
120 hpi
24 hpi
48 hpi
120 hpi
0.33 0.48
0.63 1.17*
1.41 1.93*
0.18 0.17
0.20 0.18
1.32 1.10*
Analysis of significance was calculated according to the paired sample t-test method with SPSS software (* denotes P < 0.05). a The second leaves were pre-infected with recombinant BSMV-g or BSMVTaULP5. The BSMV-TaULP5 cDNA fragment was derived from 30 UTR sequence of TaULP5. BSMV-g was the control. The fourth leaves were inoculated with Pst pathotype CYR32. hpi, hours post-inoculation. b Average size of necrotic area calculated from 45 infection sites (units in 1000 mm2, measured by DP-BSW software). c Average distance from the base of substomatal vesicles to hyphal tips calculated from 45 infection sites (units in 100 mm, measured by DP-BSW software).
regulatory role in most cellular processes [24,31]. Ubiquitin-like protein 5, as a newly characterized protein modifier in eukaryotes, has unique features and functions [29]. However, there is almost no report on UBL5 in plants, particularly its function in plant stress response. In this study, the full-length cDNA of wheat ubiquitin-like protein 5 was isolated, and TaULP5 was homologous to many sequences from monocotyledon and dicotyledon plants, even liana
and xylophyta. Human ULP5 was also highly homologous to the ULP5s from Arabidopsis, nematode, fission yeast and Saccharomyces cerevisiae [13]. Thus, we concluded that ULP5 genes are highly conserved. Meanwhile, TaULP5 was found to be localized to the cytoplasm, which was consistent with bioinformatic predictions [13]. also found that ULP5 was localized to the cytoplasm. However, the other ULPs members were demonstrated to be localized in the nucleus [6,29]. These findings indicated that ULP5 may have more unique functions than other ULPs. As an important component at translational level, the biological function of some ULPs had been explored [3]. found that SUMO-4 of human can cooperate with many clinically relevant stress-related proteins. Moreover, ULPs have been shown to modify proteins conferring functions related to programmed cell death, autophagy and regulation of the immune system [4]. In 2013, ULP5 was confirmed to be a negative regulator of tumor suppression [1]. However, no ULP5 function in plant stress response was explored. In this study, the TaULP5 expression was only largely induced in compatible interaction of APR wheat seedlings to Pst. Further VIGS analysis found that TaULP5 could contribute to the compatible interaction between XZ seedlings and Pst. In previous studies, it was found that the different interactions between XZ and Pst could be regulated by many different expressed genes [17]. Meanwhile [11], found two miRNAs could affect the compatible interaction of XZ and Pst by regulating the expression of MDHAR, an important
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member of AsA-GSH cycle. So, the interactions of XZ and Pst may be regulated by the expression transition of many genes during the different growth stages, maybe at the transcriptional level, posttranscriptional level, even translational level. In addition, Pst is a biotrophic pathogen, and it needs to maintain cell viability for disease development. The up-regulated expression of TaULP5 in the compatible interaction may prevent the occurrence of cell death to offer a suitable environment for Pst infection and development. Thus, we speculated that ULP5 may negatively regulate the cell death of host, and function as a susceptibility-related gene. Actually, the studies of susceptibilityrelated genes have opened up a new field for uncovering the mechanisms of plant-pathogen interactions [26]. found that when the PMR6 gene was knocked out, the Arabidopsis resistance to powdery mildew was increased. Meanwhile, MLO could improve plant susceptibility to powdery mildew by down-regulating the expression of PEN gene [14]. However, the detailed molecular mechanisms of TaULP5 in the interactions between XZ and Pst need to be further explored.
[8] [9]
[10]
[11]
[12] [13]
[14] [15]
[16]
5. Conclusion In the present research, the wheat ubiquitin-like 5 protein gene was isolated and confirmed to contribute to the compatible interaction of APR wheat seedlings to Pst. This research improves the understanding of ULP5 function of plant in stress response. Meanwhile, the results lay a foundation for understanding the mechanisms of different interactions between APR wheat plants and Pst at post-translational level. Acknowledgments This work was supported by grants from the National Basic Research Program of China (2013CB127700) and the 111 Project from the Ministry of Education of China (B07049).
[17]
[18]
[19]
[20]
[21] [22]
[23]
Appendix A. Supplementary data [24]
Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.pmpp.2016.06.008. [25]
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Physiological and Molecular Plant Pathology journal homepage: www.elsevier.com/locate/pmpp
TaULP5 contributes to the compatible interaction of adult plant resistance wheat seedlings-stripe rust pathogen Hao Feng a, Qiuling Wang b, Xiaoqiong Zhao b, Lina Han b, Xiaojie Wang a, Zhensheng Kang a, * a b
College of Plant Protection, State Key Laboratory of Crop Stress Biology for Arid Areas, Northwest A&F University, Yangling, Shaanxi, 712100, China College of Life Sciences, Northwest A&F University, Yangling, Shaanxi, 712100, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 March 2016 Received in revised form 16 June 2016 Accepted 27 June 2016 Available online 29 June 2016
Adult plant resistance indicates that plant is susceptible to pathogen at seedling stage, but resistant at adult stage. Understanding the mechanism of the interactions between APR wheat plants and Puccinia striiformis f. sp. tritici (Pst) is important for the creation of strategies to improve cultivar disease resistance. In this study, a full-length cDNA was isolated from APR wheat cultivar Xingzi 9104 (XZ), and was designated as ubiquitin-like protein 5 (TaULP5). TaULP5 was likely to be located in the cytoplasm, with a percentage of 75.9% Arabidopsis protoplasts number. The expression of TaULP5 was largely induced in the compatible interaction of wheat seedlings to Pst, while no obvious change was found in the incompatible interaction of wheat adult plants to Pst. Moreover, when TaULP5 was knocked down, the wheat resistance at seedling stage to Pst was improved. In addition, knockdown of TaULP5 increased the expression levels of some biotic stress-related genes, such as PR1 and PR2. It is the first time to confirm that ubiquitin-like protein could contribute to the compatible interaction of XZ to Pst, and the results will lay a foundation for understanding the mechanisms of different interactions between APR wheat plants and Pst at posttranslational level. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Abiotic stress Adult plant resistance Puccinia striiformis f. sp. tritici Ubiquitin-like protein 5 Wheat
1. Introduction Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most destructive wheat diseases in the world. Breeding and rational utilization of disease-resistant varieties is the safest, the most effective, the most economical and the most environmentally sound method for controlling wheat stripe rust [5,8]. At present, wheat resistance to Pst can be broadly categorized into allstage resistance (seedling resistance) and adult plant resistance (APR). Compared with seedling resistance, APR is much more stable and durable. APR indicates that plant is susceptible to pathogen at seedling stage, but resistant at adult stage. Understanding the
Abbreviations: dpvi, days post virus inoculation; EF, elongation factor 1 alphasubunit gene; EST, expressed sequence tag; hpi, hours post-inoculation; ORFs, Open reading frames; PcG, Polycomb group; PDS, phytoene desaturase gene; Pst, Puccinia striiformis f. sp. tritici; RT-PCR, Reverse-transcription polymerase chain reaction; UBLs, Ubiquitin-like proteins; UBP, ubiquitin-specific protease gene; ULP5, Ubiquitin-like protein 5; UPS, ubiquitin-proteasome system; VIGS, virus-induced gene silencing; XZ, Xingzi9104. * Corresponding author. E-mail address: [email protected] (Z. Kang). http://dx.doi.org/10.1016/j.pmpp.2016.06.008 0885-5765/© 2016 Elsevier Ltd. All rights reserved.
mechanisms of different interactions between APR wheat and Pst is important for the strategies creation of improving cultivar disease resistance. Post-translational modification of proteins through the ubiquitin-proteasome system (UPS) serves a critical regulatory role in most cellular processes [7,9,20,22]. Ubiquitins, as a group of highly conserved eukaryotic polypeptides 76 amino acids in length, play roles by attaching to lysine residues (or sometimes to other amino acids) of their target proteins via their C-terminal glycines [12,15]. Ubiquitin-like proteins (UBLs) are another important component of the UPS. And there is a resemblance between UBLs and ubiquitins in sequence [16]. All UBLs share a similar threedimensional structure [25]. UBLs are ligated to their target proteins or other molecules by distinct but evolutionarily related enzyme cascades [18]. Host UBL pathways have been confirmed to play important roles in plant-pathogen interactions. For example, the ubiquitin-like protein ISG15 was found to be involved in the host antiviral immune defense [24,31]. Ubiquitin-like protein 5 (ULP5) is a recently identified component of the UPS. UBL5 was first identified in 2001 as an 8.5-kD protein product of a gene isolated from human adult iris cDNAs
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[13]. Unlike other ubiquitins, ULP5 contains an ubiquitin super-fold with an electrostatic surface [23]. Meanwhile, the C-terminal peptide sequence of ULP5 is not a double glycine but a pair of tyrosines, followed with variable amino acid residues [29]. According to previous report, ULP5 may affect rat energy metabolism [2]. In addition, the ULP5 yeast ortholog ubiquitin-1 (Hub1) had been reported in Saccharomyces cerevisiae, and it was found to play a role in mRNA and pre-mRNA splicing [29]. However, the studies about the function of plant ULP5 were relatively backward. Little is known regarding the biological functions of plant ULP5, although many ULP5 genes have been predicted based on the ESTs and genomic information (NCBI) of some plants. Thus, whether ULP5 can participate in plant disease resistance, especially the interactions of APR plant and pathogen? In this study, TaULP5 was firstly isolated from APR cultivar Xingzi 9104 (XZ), and the gene function in the compatible interaction between XZ and Pst was explored. The results will lay a foundation for understanding the mechanisms of different interactions between APR wheat plants and Pst at post-translational level. 2. Materials and methods 2.1. Plant materials and treatments XZ processes APR to Pst pathotype CYR32, that means it is susceptible to CYR32 at the seedling stage but resistant at the adult stage [19]. XZ and CYR32 used in this study were obtained from the Institute of Plant Pathology, Northwest A&F University, Yangling, Shaanxi, China. Wheat cultivation and Pst inoculation were performed as described in Ref. [10]. Leaves were collected at 0, 24, 48 and 120 h post-inoculation (hpi). After collection, all samples were immediately frozen in liquid nitrogen and stored at 80 C. 2.2. RNA extraction and cDNA synthesis Extraction of total RNA was performed with TRIzol reagent (Invitrogen, Carlsbad, CA) following the manufacturer's instructions. DNase I was used to remove the genomic DNA from total RNA. The total RNA was quantitated using a NanoDrop™ 1000 spectrophotometer (Thermo Fisher Scientific, USA). Three mg of total RNA was used to synthesize the first-strand cDNA using the RT-PCR system (Promega, Madison, WI, USA) with the Oligo (dT) 18 primer according to the manufacturer's instructions. 2.3. Gene isolation and sequence characterization The sequence fragment identified from the transcriptome libraries of XZ challenged with Pst was used as a query probe to screen publically available wheat expressed sequence tag (EST) databases for the full length of target gene. Homologous wheat EST sequences were retrieved and assembled. Open reading frames (ORFs) in the assembled sequences were predicted by NCBI's ORF FINDER. Reverse-transcription polymerase chain reaction (RT-PCR) was used to amplify the full-length cDNA with specific primers (Table A.1). The PCR products were cloned into the pMD18-T Simple vector (TaKaRa Biotechnology) and sequenced with an ABI PRISM 3130xl Genetic Analyzer (Applied Biosystems, USA). The DNAMAN (version 5.2.2) program was used for phylogenetic comparison between the deduced protein sequences and the corresponding characterized proteins from other plant species. 2.4. Subcellular localization of TaULP5 The TaULP5 ORF was PCR-amplified without stop codons using specific primers with HindIII and BamHI restriction sites. The
sequence fragment was then sub-cloned into PTF486, which contains the eGFP open reading frame. The recombinant vector PTF486-TaULP5 was end-sequenced with an ABI PRISM 3130XL Genetic Analyzer (Applied Biosystems, USA). The primers used for vector construction were listed in Table A.1. Well-expanded leaves from 4 week-old Arabidopsis thaliana were used for protoplast preparation. Mesophyll protoplasts and reagents were prepared according to [30]. The PTF486-TaULP5-eGFP fusion construct was transformed into Arabidopsis thaliana mesophyll protoplasts using the method described by Ref. [30]. Protoplasts were incubated with plasmids at 25 C in the dark for 16 h with gentle swirling at 40 rpm. eGFP fluorescence images of transformed protoplasts were obtained using confocal microscopy with a Nikon PCM2000 (BioRad) laser-scanning confocal imaging system. The experiment was repeated every other day using the same batch of Arabidopsis plants for three times totally. 2.5. Quantitative RT-PCR (qRT-PCR) The expression profiles of TaULP5 and PR protein genes were determined using quantitative real-time PCR analyses with specific primers (Table A.1). These primers were first used to amplify the fragment by regular PCR. The cloned fragment was then sequenced to confirm primer specificity. The wheat translation elongation factor 1 alpha-subunit (EF) gene (GenBank Accession No. M90077) and cytoplasmatic ribosomal protein S13 gene (GenBank Accession No. AY736126) were selected as the internal reference genes for qRT-PCR analyses. Three independent biological replicates were used for each time point, as well as a non-template control, and the results were analyzed using the comparative threshold (2DDCT) method [21]. 2.6. Functional analysis of TaULP5 in response to Pst infection For virus-induced gene silencing (VIGS) vector construction, a 223-bp fragment was amplified using specific primers with PacI and NotI restriction sites. The TaPDS sequence fragment in BSMVTaPDS was replaced by the amplicon to generate the BSMV-TaULP5 vector. The recombinant vectors were end-sequenced with an ABI PRISM 3130XL Genetic Analyzer (Applied Biosystems, USA). The primers used for vector construction were listed in Table A.1. The detailed VIGS procedures were performed as described by Ref. [27] with a minor revision. In vitro transcription products of the three BMSV genome sequences (a, b, g) were diluted 30 times, and 0.5 mL of each was mixed in a 1:1:1 ratio. Next, 9 mL of FES buffer was added to the mixture. And then the mixtures were directly applied to the second leaves by rub inoculation with a gloved finger. BSMVTaPDS was used to silence the wheat phytoene desaturase gene (PDS) as a positive control and FES buffer was used as a negative control. The wheat seedlings inoculated with BSMV were incubated in a growth chamber at 25 ± 2 C. Nine days post inoculation (dpi), the base of the third leaves and the top of the fourth leaves were infected with CYR32. After Pst inoculation, the wheat seedlings were incubated in a growth chamber at 16 ± 2 C. The Pst-inoculated leaves were then sampled at 0, 24, 48, 120 hpi for silencing efficiency analysis and histological observation. Pst infection types were examined at 15 dpi. Two independent biological replicates were performed for each treatment. 2.7. Histological observation The samples were stained as previously described [28], and the stained leaf segments were observed to analyze the infection site, necrotic area, and hyphal length using an Olympus BX-51 microscope (Olympus Corp., Tokyo). No more than 5 different infection sites were examined on each randomly selected leaf segment, and a
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total of 45 infection sites were examined per treatment. Statistical analysis was performed with SPSS (Statistical Package for the Social Sciences) software. 3. Results 3.1. The identification of TaULP5 The sequence fragment identified from the transcriptome libraries of XZ challenged with Pst (data not publihsed) was used as a query to screen wheat EST database for full-length gene sequence.
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A 292-bp wheat cDNA sequence was identified in silico and amplified by RT-PCR; this sequence contains an open reading frame encoding a 73-amino acid polypeptide (Fig. 1a). Analysis of a multisequence alignment showed highly conservation among the deduced protein and its homologs from other plants. Moreover, the C-terminus of the deduced peptide sequence is a pair of tyrosines, which is the typical characteristic of ULPs (Fig. A.1). In addition, the phylogenetic analysis showed that the candidate gene was clustered with the ubiquitin-like protein 5 genes from Brachypodium sylvaticum and Hordeum vulgare (Fig. 1b). Therefore, we designated the candidate gene as wheat ubiquitin-like protein 5 (TaULP5).
Fig. 1. Isolation of TaULP5 and characterization of the deduced amino acid sequence. (a) The cDNA fragment of TaULP5 was identified by in silico cloning and sequencing. The longest ORF was 222 bp, encoding a protein of 73 amino acids. The underlined sequences indicate gene-specific primers for RT-PCR, and the arrows indicate the direction of amplification. The triple bases in the grey boxes are the start and stop codons. (b) A phylogenetic tree of TaULP5 constructed with the multiple alignment program DNAMAN. The branches are labeled with the gene names and GenBank accession numbers from different plant species. The GenBank accession numbers are as follows: wheat XZ, KJ476506; Arabidopsis thaliana, NP_190104.1; Brachypodium distachyon, XP_003578938.1; Cucumis sativus, XP_004139444.1; Glycine max, XP_003535365; Hordeum vulgare, ABO42268.1; Medicago truncatula, XP_003601666.1; Oryza sativa, NP_001176788.1; Theobroma cacao, XP_007016757.1; Vitis vinifera, XP_002280046.1; Zea mays, NP_001150362.1.
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3.2. TaULP5 is localized to the cytoplasm To verify the subcellular localization of TaULP5, the TaULP5 gene was fused to green fluorescent protein (eGFP). The fusion vector PTF486-TaULP5-eGFP and the control vector PTF486-eGFP were transformed into Arabidopsis thaliana mesophyll protoplasts. Fluorescence microscopic observation of transformed cells showed that the green fluorescent of 85.7% control protoplasts was distributed in the whole cell of control protoplasts, including the cytoplasm and nucleus; and the TaULP5-eGFP fusion protein was likely to be accumulated in the cytoplasm, with a percentage of 75.9% protoplasts number (Fig. 2). 3.3. TaULP5 is highly expressed in the compatible wheat-Pst interaction To investigate the function of TaULP5 in APR wheat response to
Pst, the transcript profile was evaluated by qRT-PCR in both XZ seedlings and adults challenged with Pst using two inner reference genes. TaULP5 was stably expressed with no significant changes from 24 to 120 hpi compared to the control (0 hpi) in the adult stage. However, expression of TaULP5 was significantly upregulated in XZ seedlings response to Pst (Fig. 3). 3.4. Knocking down of TaULP5 improves the resistance of XZ seedlings to Pst To identify the function of TaULP5 in the compatible interaction between XZ seedlings and Pst, a barley stripe mosaic virus (BSMV)based VIGS system was used to silence the expression of TaULP5. All BMSV-inoculated plants displayed mild chlorotic mosaic symptoms at 7 days post virus inoculation (dpvi), and no obvious defect in leaf growth was observed (Fig. 4b). Typical photobleaching phenotypes occurred on BSMV-TaPDS-inoculated plants at 12 dpvi (Fig. 4c). The
Fig. 2. Detection and subcellular localization of the TaULP5-eGFP fusion protein in mesophyll protoplasts of Arabidopsis. (a) Vector indicates the positive control, and the GFP signal was detected throughout the cell, including the nucleus. ULP represents a fusion protein, which was located in the cytoplasm. W: image of the protein under white light; M: image of the fusion protein under BW fluorescence. The GFP signal is indicated in green, and chlorophyll autofluorescence is show in red; GFP: image of the fusion protein under GW light. The GFP signal is indicated in green. Bar ¼ 10 mm. (b) Quantitation of the nucleus-versus-cytoplasm partition of the TaULP5-eGFP fusion protein as shown in (a). ‘Cytoplasm’ indicates the GFP signal was mainly located in cytoplasm, ‘nucleus-cytoplasm’ indicates the GFP signal could be observed in both nucleus and cytoplasm obviously, and ‘other’ means no typical location signal was observed.
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Fig. 3. qRT-PCR expression profiles of TaULP5 in wheat leaves in response to Pst pathotype CYR32. qRT-PCR data were normalized to the expression level of the wheat elongation factor 1 alpha-subunit (EF) gene (a) and cytoplasmatic ribosomal protein S13 (b). The relative expression levels of TaULP5 at different growth stages of plants challenged with Pst were calculated using the comparative threshold (2DDCT) method. The mean values and standard deviations were calculated with data from three independent replicates.
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infected with BSMV-TaULP5. Although the phenotype of TaULP5silenced wheat plants was still considered to be susceptible, the resistance level was obviously increased (f). To confirm whether the phenotypic changes were caused by the silence of TaULP5, qRT-PCR was used to examine the relative transcript level of TaULP5. Compared with BMSV-g-inoculated wheat leaves, the expression of TaULP5 was reduced 89%, 84% and 85% at 24, 48 and 120 hpi, respectively (Fig. 5a). Moreover, the transcript levels of four PR genes (PR1, PR2, PR3 and PR5) were also detected in TaULP5-silenced plants after inoculated with Pst. The transcript levels of TaPR1 and TaPR2 were up-regulated in TaULP5-knockdown leaves at 24 hpi, for approximately 2.6 and 6.0 fold increased, respectively. At 48 hpi, these two PR genes showed similar expression levels with BMSV-g inoculated wheat leaves. However, the expression of TaPR3 and TaPR5 showed no significant change (Fig. 5b). To further explore the enhanced stripe rust fungus resistance of TaULP5-silenced wheat leaves, hyphal growth and cell death were examined at 24, 48 and 120 hpi microscopically. As shown in Fig. 6 and Table 1, hyphal length showed no significantly difference at 24 and 48 hpi compared with that of the mock-inoculated and BSMVg-infected plants. In contrast, hyphae length in TaULP5-silenced wheat leaves was significantly shorter (P < 0.05) at 120 hpi. Moreover, larger necrotic areas around the Pst infection sites were observed at 48 and 120 hpi in the TaULP5-silenced plants. It is likely that the suppression of TaULP5 may facilitate host cell death, and hyphal growth of stripe rust pathogen was suppressed by the resistant reaction of plant.
4. Discussion Plant diseases are a major factor restricting food production worldwide. Rational utilization of APR is much more stable and durable for disease control. Thus, exploring the molecular mechanisms of APR plant-pathogen interactions is necessary for the generation of new disease-control strategies. Previous studies have found that ubiquitin-proteasome system (UPS) plays a critical
Fig. 4. Phenotype of TaULP5-knockdown leaves from Pst-challenged wheat plant seedlings. For functional analysis of TaULP5, virus-induced gene silencing (VIGS) was applied to seedling-stage wheat plants. No change was found in the plant preinoculated with FES buffer (a). All of the BSMV-g-infected plants displayed mild chlorotic mosaic symptoms at 7 days post virus inoculation (dpvi) in the seedling stage (b). Photobleaching was observed at 12 dpvi when the leaves were inoculated with the positive control combination of BSMV-TaPDS vectors (c). A typical phenotype was observed when leaves pre-inoculated with FES buffer and BSMV-g were challenged with CYR32 (d and e). Phenotypic changes were observed in TaULP5-knockdown leaves (f) at 14 days post-inoculation of Pst.
leaves of the mock-inoculated plants and those infected with BSMV-g and BSMV-TaULP5 were further inoculated with CYR32. Fourteen days later, a fully compatible phenotype was observed on leaves of the plants mock inoculated with FES buffer and infected with BSMV-g (Fig. 4d and e). However, except for slight sporulation, typical necrotic spots emerged on leaves of the plants previously
Fig. 5. Silencing efficiency of TaULP5 and PR proteins expression in TaULP5knockdown leaves as measured by qRT-PCR. The silencing efficiency of TaULP5 (a) and the relative expression levels of PR proteins (b) in TaULP5-knockdown leaves challenged with pathotypes CYR32 were calculated using the comparative threshold (2DDCT) method. The data were normalized to the wheat elongation factor 1 alphasubunit (EF) expression level. Mean values and standard deviations were calculated with data from two independent replicates.
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Fig. 6. Histological observation of host cells and pathogen hyphal change in the TaULP5-knockdown leaves responding to Pst. The TaULP5-knockdown leaves and control leaves challenged with Pst pathotype CYR32 were sampled at 24, 48, and 120 hpi. The hyphae were stained to facilitate analysis of the length change between control leaves (A, B, C) and TaULP5-knockdown leaves (a, b, c). The histological changes were observed under an epifluorescence microscope. D-F and d-f indicate the change in necrotic cell area at 24, 48 and 120 hpi in the control and TaULP5-knockdown leaves, respectively. Bar ¼ 20 mm. HMC, haustorial mother cell; IH, initial hypha; NC, necrotic cells; SH, second hypha; SV, substomatal vesicle.
Table 1 Histological analysis of wheat TaULP5 knockdown leaves responding to Pst pathotype CYR32*. Treatmenta
BSMV-g BSMV-TaULP5
Necrotic areab
Hyphal lengthc
24 hpi
48 hpi
120 hpi
24 hpi
48 hpi
120 hpi
0.33 0.48
0.63 1.17*
1.41 1.93*
0.18 0.17
0.20 0.18
1.32 1.10*
Analysis of significance was calculated according to the paired sample t-test method with SPSS software (* denotes P < 0.05). a The second leaves were pre-infected with recombinant BSMV-g or BSMVTaULP5. The BSMV-TaULP5 cDNA fragment was derived from 30 UTR sequence of TaULP5. BSMV-g was the control. The fourth leaves were inoculated with Pst pathotype CYR32. hpi, hours post-inoculation. b Average size of necrotic area calculated from 45 infection sites (units in 1000 mm2, measured by DP-BSW software). c Average distance from the base of substomatal vesicles to hyphal tips calculated from 45 infection sites (units in 100 mm, measured by DP-BSW software).
regulatory role in most cellular processes [24,31]. Ubiquitin-like protein 5, as a newly characterized protein modifier in eukaryotes, has unique features and functions [29]. However, there is almost no report on UBL5 in plants, particularly its function in plant stress response. In this study, the full-length cDNA of wheat ubiquitin-like protein 5 was isolated, and TaULP5 was homologous to many sequences from monocotyledon and dicotyledon plants, even liana
and xylophyta. Human ULP5 was also highly homologous to the ULP5s from Arabidopsis, nematode, fission yeast and Saccharomyces cerevisiae [13]. Thus, we concluded that ULP5 genes are highly conserved. Meanwhile, TaULP5 was found to be localized to the cytoplasm, which was consistent with bioinformatic predictions [13]. also found that ULP5 was localized to the cytoplasm. However, the other ULPs members were demonstrated to be localized in the nucleus [6,29]. These findings indicated that ULP5 may have more unique functions than other ULPs. As an important component at translational level, the biological function of some ULPs had been explored [3]. found that SUMO-4 of human can cooperate with many clinically relevant stress-related proteins. Moreover, ULPs have been shown to modify proteins conferring functions related to programmed cell death, autophagy and regulation of the immune system [4]. In 2013, ULP5 was confirmed to be a negative regulator of tumor suppression [1]. However, no ULP5 function in plant stress response was explored. In this study, the TaULP5 expression was only largely induced in compatible interaction of APR wheat seedlings to Pst. Further VIGS analysis found that TaULP5 could contribute to the compatible interaction between XZ seedlings and Pst. In previous studies, it was found that the different interactions between XZ and Pst could be regulated by many different expressed genes [17]. Meanwhile [11], found two miRNAs could affect the compatible interaction of XZ and Pst by regulating the expression of MDHAR, an important
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member of AsA-GSH cycle. So, the interactions of XZ and Pst may be regulated by the expression transition of many genes during the different growth stages, maybe at the transcriptional level, posttranscriptional level, even translational level. In addition, Pst is a biotrophic pathogen, and it needs to maintain cell viability for disease development. The up-regulated expression of TaULP5 in the compatible interaction may prevent the occurrence of cell death to offer a suitable environment for Pst infection and development. Thus, we speculated that ULP5 may negatively regulate the cell death of host, and function as a susceptibility-related gene. Actually, the studies of susceptibilityrelated genes have opened up a new field for uncovering the mechanisms of plant-pathogen interactions [26]. found that when the PMR6 gene was knocked out, the Arabidopsis resistance to powdery mildew was increased. Meanwhile, MLO could improve plant susceptibility to powdery mildew by down-regulating the expression of PEN gene [14]. However, the detailed molecular mechanisms of TaULP5 in the interactions between XZ and Pst need to be further explored.
[8] [9]
[10]
[11]
[12] [13]
[14] [15]
[16]
5. Conclusion In the present research, the wheat ubiquitin-like 5 protein gene was isolated and confirmed to contribute to the compatible interaction of APR wheat seedlings to Pst. This research improves the understanding of ULP5 function of plant in stress response. Meanwhile, the results lay a foundation for understanding the mechanisms of different interactions between APR wheat plants and Pst at post-translational level. Acknowledgments This work was supported by grants from the National Basic Research Program of China (2013CB127700) and the 111 Project from the Ministry of Education of China (B07049).
[17]
[18]
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Appendix A. Supplementary data [24]
Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.pmpp.2016.06.008. [25]
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