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Journal of Integrative Agriculture 2016, 15(6): 1293–1303
RESEARCH ARTICLE
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An improved method for RNA extraction from urediniospores of and wheat leaves infected by an obligate fungal pathogen, Puccinia striiformis f. sp. tritici
MA Li-Jie1, 3, QIAO Jia-xing1, KONG Xin-yu1, WANG Jun-juan1, XU Xiang-ming1, 2, HU Xiao-ping1
1 State Key Laboratory of Crop Stress Biology for Arid Areas, College of Plant Protection, Northwest A&F University, Yangling 712100, P.R.China
2 Genetics and Crop Improvement, East Malling Research, East Malling ME19 6BJ, UK3 Erdos Ecological Environment of Career Academy, Ordos 017010, P.R.China
AbstractStripe rust, caused by Puccinia striiformis f. sp. tritici, is an important wheat disease in China, seriously threatening wheat production. Understanding the winter survival of the fungus is a key for predicting the spring epidemics of the disease, which determines the crop loss. Estimation of P. striiformis f. sp. tritici winter survival requires processing a large number of samples for sensitive detection of the pathogen in wheat leaf tissue using real-time quantitative reverse transcription PCR (qRT-PCR). A bottleneck for the analysis is the acquisition of a good yield of high quality RNA suitable for qRT-PCR to distinguish dead and alive fungal hyphae inside leaves. Although several methods have been described in the literatures and commercial kits are available for RNA extraction, these methods are mostly too complicated, expensive and inefficient. Thus, we modified three previously reported RNA extraction methods with common and low-cost reagents (LiCl, SDS and NaCl) to solve the problems and selected the best to obtain high quality and quantity RNA for use in qRT-PCR. In the three improved methods, the NaCl method was proven to be the best for extracting RNA from urediniospores of and wheat leaves infected by P. striiformis f. sp. tritici, although the modified LiCl and SDS methods also increased yield of RNA compared to the previous methods. The improved NaCl method has the following advantages: 1) Complete transfer of urediniospores of P. striiformis f. sp. tritici from the mortar and pestle can ensure the initial amount of RNA for the qRT-PCR analysis; 2) the use of low-cost NaCl to replace more expensive Trizol can reduce the cost; 3) the yield and quality of RNA can be increased; 4) the improved method is more suitable for a large number and high quantity of samples from fields. Using the improved NaCl method, the amount of RNA was increased three times from urediniospores of P. striiformis f. sp. tritici compared from the extraction kit. Approximately, 10.11 μg total RNA of high quality was obtained from 100 mg of infected leaves, which was 8.8, 6.5, 3.4 and 2.1 folds of the amounts obtained from the previous LiCl, SDS, NaCl and traditional Trizol methods, respectively. The method could be used to study the overwintering rates of P. striiformis f. sp. tritici over a large region of
Received 7 September, 2015 Accepted 15 December, 2015MA Li-jie, E-mail: [email protected]; Correspondence HU Xiao-ping, Tel/Fax: +86-29-87091095, E-mail: [email protected]
© 2016, CAAS. Published by Elsevier Ltd. This is an open access art ic le under the CC BY-NC-ND l icense (http:/ /creativecommons.org/licenses/by-nc-nd/4.0/).doi: 10.1016/S2095-3119(15)61250-3
1294 MA Li-Jie et al. Journal of Integrative Agriculture 2016, 15(6): 1293–1303
1. Introduction
Stripe rust, caused by Puccinia striiformis Westend. f. sp. tritici Erikss., is one of the most destructive diseases of wheat worldwide (Chen 2005). Spring is the most destruc-tive period in epidemic of wheat stripe rust, and the disease severity is mainly determined by the level and distribution of overwintered pathogen and the climate conditions during the spring (Li and Zeng 2002; Chen 2005). Under natural conditions, P. striiformis f. sp. tritici begins to overwinter as mycelium in host plant tissue once temperature declines to 1–2°C (Li and Zeng 2002), and remains alive in plant tissue as mycelium for months when temperatures are between about 2 and –15°C (Li and Zeng 2002; Sharma-Poudyal and Chen 2009). Wheat plants infected by P. striiformis f. sp. tritici before the winter often remain symptomless until the weather becomes warm. Field investigation of wheat stripe rust is conventionally conducted through visual obser-vations, which could not detect the pathogen in plant tissue before sporulation. For fields even known to be infected before the winter, visual observations could not determine whether the pathogen is alive or dead. To better understand the pathogen survival, the real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) tech-nique has been used for determining P. striiformis f. sp. tritici survival rates in different periods and regions. qRT-PCR has been extensively applied in detecting and quantifying animal and human pathogens since the first instrument was commercialized in 1997 (De Francesco 2003). For plant pathogens, quantitative real-time PCR (qPCR) has been used for quantifying fungal genomic DNA from soil substrates (Filion et al. 2003), estimating infection rates of wheat seeds by Tilletia caries (McNeil et al. 2004), and detecting airborne spore density of Monilinia fructicola in stone fruit orchards (Luo et al. 2007). qPCR has also been used to quantify P. striiformis f. sp. tritici inside wheat leaves during latent infections (Pan et al. 2010; Yan et al. 2011). Because RNA existed in living biomass could represent alive pathogens, qRT-PCR assay, which uses RNA, should be more useful than qPCR, which uses DNA, for quantification of alive pathogens.
A bottleneck for qRT-PCR studies is the acquisition of sufficient quantities of high quality RNA from a large number of samples in a timely and cost-effective manner
(Leite et al. 2012). Currently, physical treatment such as freezing the materials with liquid nitrogen and grinding with in a mortar with a pestle is the most commonly used meth-od (Peng et al. 2007). Because P. striiformis f. sp. tritici is very difficult to culture on a medium, it is relatively hard to obtain a large quantity of urediniospores compared to many other filamentous fungi. Moreover, P. striiformis f. sp. tritici urediniopsores have thick cell wall (Kang et al. 1993) which is hard to break. When urediniospores are ground in liquid nitrogen in a mortar with a pestle, it is very hard to scrape the ground powdery out from the mortar and pestle. It is also possible to splash samples when adding liquid nitrogen and during the grinding. All of these problems can result in less ground powder, which may not represent the initial quantity of the pathogen RNA in a sample and may increase artificial variations among samples.
So far, many methods have been published for isolating RNA. However, only few are available for isolating RNA from urediniospores and wheat leaves infected by P. striiformis f. sp. tritici. Total RNA extraction from urediniospores of P. striiformis f. sp. tritici was studied by liquid nitrogen grind-ing combining with an RNA isolation kit (Peng et al. 2007). The methods of LiCl, SDS and NaCl were also used to ex-tract total RNA from wheat leaves infected by P. striiformis f. sp. tritici (Cui et al. 2006; Yu et al. 2007). However, these methods are too complicated, expensive, time-consuming, and/or with a low efficiency. Therefore, a better method which is more simple, less expensive, higher efficient, and more stable is needed.
The objective of this study was to improve RNA extraction methods for extracting high-quality and large-quantity of total RNA from urediniospores and wheat leaves infected by P. striiformis f. sp. tritici to be used in RT-PCR and qRT-PCR assays.
2. Results
2.1. A new procedure for fully transferring materials from mortars and tubes
When the previously described methods (Cui et al. 2006; Yu et al. 2007) were used, urediniodpores of P. striiformis f. sp. tritici adhered to the wall of centrifuge tube (Fig. 1-B, left), and the ground spore powdery was difficult to scrape out entirely from the mortar and pestle (Fig. 1-A, left). The yield of total RNA from fresh urediniospores was low because
wheat production for predicting epidemic levels by determining pathogen survival levels after winter. The method can also be used in any studies which need a large number of high quality RNA samples.
Keywords: Puccinia striiformis f. sp. tritici, RNA extraction, RT-PCR, urediniospore, mycelium
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of partial loss during the grinding and transferring process (Table 1). In contrast, little powdery residue was found in the mortars (Fig. 1-A, right) and tubes (Fig. 1-B, right) when 40 μL Tris-HCl (pH 8.0) mixing with 10 mg material was added to the procedures of the three methods, which helped completely break cell wall and detach the spore powder from the mortar and pestle. The yield of total RNA from urediniospores treated with Tris-HCl (pH 8.0) was nearly tripled compared with urediniospores alone (Table 1). This improvement ensured that ground powdery could represent initial material quality and could finally contribute to the quantity of RNA for qRT-PCR. Before this procedure was established, several other methods were tried to overcome the problem. Ground acid-washed quartz sand was found to be able to mix well with urediniospores and resulted in no-residual either in the mortars or tubes. However, the lower value than the standard OD260/230 ratio (Table 1) indi-
cated relatively high polysaccharide or polyphenol contam-ination. Furthermore, the total RNA obtained was seriously degraded, indicated by agarose gel electrophoresis (data not shown), although the quantity of RNA was higher than the urediniospore alone method. The obtained RNA was not pure enough for use in qRT-PCR. RNA extracted using RNA-free water was also not as good as that using the Tris-HCl method (Table 1).
2.2. Integrated analysis of RNA extracted using the three improved methods
The integrity of total RNA from urediniospores (Fig. 2-A) and wheat leaves (non-inoculation and inoculation) (Fig. 2-C) using the three improved methods (LiCl, SDS and NaCl) were determined. All RNA samples could be successfully reverse-transcripted into cDNA and were detected using PCR with specific primers EF1-F and EF1-R of P. strii-formis f. sp. tritici (Fig. 2-B and D). For urediniospores, the quantity of purified RNA from 5 mg urediniospores was 126.98 ng μL–1 (LiCl), 333.10 ng μL–1 (SDS) and 253.17 ng μL–1 (NaCl), respectively. For wheat leaves, collected from healthy (non-inoculated) and diseased (inoculated with P. striiformis f. sp. tritici 9 days post inoculation (dpi)), the yield of total RNA from healthy wheat leaves were about three times of the diseased wheat leaves no matter what crude or purified RNA and the extraction methods were used (Table 2). Using the improved methods, the puri-fied RNA quantity lost was ~8% less than the crude RNA (Table 2). The yield of total crude or purified RNA obtained using the improved NaCl method was the highest among the three methods for both healthy and diseased leaves. The repeatedly obtained OD260/230 ratios greater than 2.0 indicated that there was no detectable polysaccharide and polyphenol contamination. However, the OD260/280 ratios were between 2.1 and 2.3, greater than 2.0, which might be caused by nucleic acid hydrolysis into nucleotide and/or adding Tris-HCl (pH 8.0).
Total RNA from 1, 5, 10, 20 and 30 mg urediniospores were extracted using the three improved methods. All total RNA had good integrity in different quantities of ured-iniospores (Fig. 3). The improved SDS method was more
A
B
Fig. 1 A new procedure was used in transferring urediniospores of Puccinia striiformis f. sp. tritici from mortars and tubes. A, the residues of urediniospore powder were remained in the mortar after grinding (left). Little residue was found in the mortar using the improved method (right). B, transferring urediniospores after weighing. Urediniospores adhered to the inner-wall of the tube (left), and little residue was remained in the inner-wall of the tube (right).
Table 1 Comparison of the amounts of purified total RNA extracted using different treatments during grinding in liquid nitrogen
TreatmentPurified total RNA
Concentration (ng μL–1) OD260/280 OD260/230
Urediniospores 81.33±12.41 b 1.92±0.02 a 2.01±0.01 aUrediniospores & RNA-free water 121.48±5.94 b 1.99±0.02 ab 2.07±0.04 aUrediniospores & Tris-HCl (pH 8.0) 221.45±14.98 a 2.01±0.01 ab 2.12±0.01 aUrediniospores & quartz sand 188.93±11.52 a 2.08±0.02 a 0.83±0.07 bThe values were the averages of three replicates, and the value following by “±” was standard error. Values in the same column marked with different letters were significantly different (P=0.05) in Tukey test of one-way ANOVA. The same as below.
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suitable for low quantities of material, especially less than 15 mg, than the improved NaCl and LiCl methods (Fig. 4). In contrast, the improved NaCl method was better for ex-tracting RNA from a higher quantity of material (over 15 mg) than the improved LiCl and SDS methods. The results of the LiCl method were close to the NaCl method when the material quantity was higher than 15 mg, but lower than the NaCl method with material quantity of 15 mg or less (Fig. 4). The improved NaCl method demonstrated a relatively high extraction efficiency (R2=0.999) compared to the improved LiCl (R2=0.996) and SDS (R2=0.926) methods (Fig. 4). So, the improved NaCl method is more useful for extracting large-scale weight of materials, such as wheat leaves over 100 mg.
Using the improved NaCl method, approximately 10.11 μg
(Table 3) total RNA of high purity was obtained from 100 mg of wheat leaves infected by P. striiformis f. sp. tritici 9 dpi, and was approximately 8.8, 6.5 and 3.4 folds of the three unim-proved methods of LiCl, SDS (Yu et al. 2007) and NaCl (Cui et al. 2006), respectively. It was 2.1 folds of the traditional Trizol method (Yu et al. 2007). Using the improved LiCl method, the extracted total RNA was 9.33 μg, approximately 8.1 folds of the unimproved method. The improved SDS method was nearly 4.6 folds of the unimproved method. For urediniospores, 24.4 μg (NaCl), 23.59 μg (LiCl) and 18.19 μg (SDS) of total RNA was respectively routinely obtained from 30 mg of fresh urediniospores using the improved methods. For both urediniospores and wheat leaves, the improved NaCl method was proven to be the best among the three methods.
A B
C E
1 2 3 1 2 3M CK
h1 h2 h3 d1 d2 d3 M CK h1 h2 h3 d1 d2 d3
D
Fig. 2 Total RNA extracted with different methods from urediniospores and wheat leaves infected by P. striiformis f. sp. tritici. A, total RNA was separated on 1% agarose gel containing EtBr and photographed under ultraviolet light. Lanes 1, 2 and 3 are total RNA of 5 mg urediniospores extracted using the LiCl, SDS and NaCl methods, respectively. B, RNA products of RT-PCR for marker (159 bp) using a pair of primers for EF1 of P. striiformis f. sp. tritici. C and D, the total RNA of healthy wheat leaves (non-inoculated) and diseased wheat leaves (inoculated with P. striiformis f. sp. tritici) extracted using the improved LiCl, SDS and NaCl methods, respectively. E, the RNA products of RT-PCR amplified using a pair of primers for EF1 of P. striiformis f. sp. tritici (159 bp). M, DNA marker (600, 500, 400, 300, 200, and 100 bp); CK, negative control with template of distilled water; h, heathy wheat leaves; d, diseased wheat leaves.
Table 2 Comparison of three methods for RNA extraction from healthy and infected wheat leaves by Puccinia striiformis f. sp. tritici
Treatment Method Weight(mg)
Crude RNA Purified RNAConcentration (ng μL–1) OD260/280 OD260/230 Concentration (ng μL–1) OD260/280 OD260/230
Healthy leaves
LiCl 300.3 8 361.01±788.21 ab 2.13±0.01 a 2.23±0.02 a 8 036.52±185.86 b 2.21±0.03 a 2.32±0.04 aSDS 300.2 6 604.65±383.41 b 2.26±0.04 a 2.36±0.01 a 5 986.91±257.09 c 2.22±0.02 a 2.36±0.01 aNaCl 299.8 9 981.82±10.50 a 2.19±0.05 a 2.26±0.06 a 9 213.53±22.87 a 2.20±0.01 a 2.26±0.03 a
Infected leaves
LiCl 300.1 2 631.23±93.28 a 2.25±0.03 a 2.06±0.04 a 2 400.23±118.15 a 2.16±0.06 a 2.16±0.05 aSDS 299.9 2 477.86±88.93 a 2.17±0.05 a 2.26±0.03 a 2 285.02±58.87 a 2.14±0.01 a 2.08±0.04 aNaCl 300.2 2 833.20±62.14 a 2.22±0.03 a 2.37±0.01 a 2 616.91±51.27 a 2.20±0.06 a 2.25±0.02 a
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2.3. The improved NaCl method was successfully used in RT-PCR with inoculated and non-inoculated wheat leaves from the field
Wheat leaves of different disease developmental stages (latent, initial symptom-expression, mid-sporulation and
late sporulation) after inoculation were collected from the field. The total RNA was extracted from the samples using the improved NaCl method. Intact RNA was extracted from latent and initial symptom-expression samples, while the RNA from the later stages (mid-sporulation and late-sporu-lation) was degraded (Fig. 5). The P. striiformis f. sp. tritici hyphae inside the leaves could be easily detected, even for latent leaves which could not be counted as diseased using traditional field investigation (Fig. 5). The yields of RNA from four different stages (latent, initial symptom-expression, mid-sporulation and late-sporulation) were different. The quantity of RNA in the latent stage was the highest and in the initial symptom-expression stage was the lowest (Fig. 6).
3. Discussion
Obtaining adequate and pure RNA is a critical step in a successful study to monitor the survival rate of overwintered pathogens, such as P. striiformis f. sp. tritici, for accurately forecasts of the diseases using qRT-PCR. In this study, we improved methods for extracting RNA with high quantity and quality from urediniospores and wheat leaves infected by P. striiformis f. sp. tritici, and used the best improved method (the NaCl method) to detect P. striiformis f. sp. tritici
A B C
D E F
1 2 3 1 2 3 1 2 3 1 2 3
1 2 3 1 2 3 1 2 3 1 2 3
1 2 3 1 2 3
Fig. 3 Three different methods were used for extracting RNA from urediniospores of P. striiformis f. sp. tritici. Total RNA was separated on 1% agarose gel containing EtBr and photographed under ultraviolet light. A, B, C, D and E, RNA extracted from 1, 5, 10, 20 and 30 mg of urediniospores, respectively. F, the clean mortars after grinding and transferring the material. Lanes 1, 2 and 3 in the left, crude RNA extracted using the improved LiCl, SDS and NaCl methods, respectively. Lanes 1, 2 and 3 in the right, purified RNA after extracted using the improved LiCl, SDS and NaCl methods, respectively.
0
400
800
1 200
1 600
2 000
0 5 10 15 20 25 30
Con
cent
ratio
n (n
g μL
–1)
Weight (mg)
LiCl-crude RNALiCl-purified RNASDS-crude RNASDS-purified RNANaCl-crude RNANaCl-purified RNA
Fig. 4 Comparison of the improved LiCl, SDS and NaCl methods for total RNA extracted from urediniospores of P. striiformis f. sp. tritici with 1, 5, 10, 20 and 30 mg, respectively. The solid and dotted lines represent purified and crude RNA, respectively.
Table 3 Comparison of different total RNA quantities from urediniospores of and wheat leaves infected by P. striiformis f. sp. tritici
Treatment Weight(mg)
Total RNA quantity for different extraction methods (μg)Reference method Improved method
LiCl SDS NaCl Trizol Isolation kit LiCl SDS NaClInfected leaves 100 1.15 1.56 3.00 4.72 – 9.33±0.77 ab 7.23±0.19 b 10.11±0.75 aUrediniospores 30 – – – – 7.50 23.59±1.03 a 18.19±0.04 b 24.40±0.11 a
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in inoculated wheat plants from a field plot. The method has several advantages over the traditional methods. The most remarkable advantage is the overcoming of the prob-lem of losing material caused by ground powdery sticking to mortars and splashing out during adding liquid nitrogen and grinding. Using the improved methods, ground powder representing the initial material for qRT-PCR analysis could
be obtained from various materials, such as urediniospores, wheat leaves, etc. We attempted to use quartz sand, talcum powder or diatomite (data not shown) for mixing with ured-iniospores. Although the acquired powder was completely transferred from mortar and tube, the total RNA obtained was seriously degraded. Some of the substances, such as MgO2, Al2O3, and Fe2O3, may damage RNA. We also
Latent Initial symptom-expression Mid-sporulation Late-sporulation
l1 l2 l3 n1 n2 n3 m1 m2 m3 a1 a2 a3
l1 l2 l3 n1 n2 n3 m1 m2 m3 a1 a2 a3 rtl rtn rtm rta rtCKCKM
Fig. 5 The improved NaCl method was used in extracting total RNA of wheat leaves of different disease developmental stages after inoculated with P. striiformis f. sp. tritici. Lanes 1, 2 and 3 represent three repeats for each of the four disease developmental stages (l=latent, n=initial symptom-expression, m=mid-sporulation, a=late-sporulation). A pair of primers for EF1 of P. striiformis f. sp. tritici was used to detect RNA through RT-PCR (159 bp). Lanes rtl, rtn, rtm and rta were negative controls without including enzyme mixture for RT-PCR using different templates (latent, initial symptom-expression, mid-sporulation and late-sporulation), respectively. Lane rtCK was negative control without including template during the RT-PCR.
0100200300400500600700800900
1 000
Latent Initial symptom-expression
Mid-sporulation Late-sporulation
Con
cent
ratio
n (n
g μL
–1)
Stages
Crude RNA Purified RNAa
a
bb
cc
cc
Fig. 6 Comparison of the quantities of total RNA extracted from four stages of wheat stripe rust in field using the improved NaCl method. Bars in the same pattern marked with different letters were significantly different (P=0.05) in Turkey test of one-way ANOVA.
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attempted to replace Tris-HCl (pH 8.0) with RNA-free water or RNA extracting solution, but the results were not as good as the method using Tris-HCl.
Three common RNA extraction methods (LiCl, SDS and NaCl) were used in the improvement study because the materials are relatively cheap. The cost is a common consideration when a large number of samples are needed in a study. All of the three methods were modified for fully transferring materials to reduce material lost and shortening the extraction process to reduce contamination and RNA lost. We also improved several steps for sufficiently breaking urediniospore cell wall, preventing oxidation and removing proteins. In the LiCl method (Yu et al. 2007), 2 volume of chloroform:Isoamyl alcohol (49:1) was added to reduce protein contamination, and the RNA suspension was precip-itated again with 600 μL buffer (2 mol L–1 LiCl and 50 mmol L–1 EDTA) after first precipitatation with isopropyl alcohol. While in the modified LiCl method, 750 μL phenol:chloro-form:isoamyl alcohol (25:24:1) was added to enhance the ability of reducing protein contamination. Phenol can be used to remove more proteins than chloroform, but phenol can easily remain in extracted RNA, but can be removed in a second extraction using chloroform, as shown in this study. To purify RNA, isopropyl alcohol was used to precipitate RNA, and the results were better than before. In the SDS method (Yu et al. 2007), 16% PVP was not added to resist oxidation. In the whole extraction process, the extraction of four times in the previous study (two times used in crude RNA by phenol:chloroform:isoamyl alcohol (25:24:1) and other two times used in purified RNA by phenol:chloro-form:isoamyl alcohol (25:24:1) and chloroform) could take a lot of time and also lose a lot of RNA. In the improved SDS method, twice extraction was used in the process of crude RNA using phenol:chloroform:isoamyl alcohol (25:24:1) and chloroform, respectively. Protein contamination was removed through two extractions (phenol and chloroform). In the modified NaCl method, 1% β-mercaptoethanol and 16% PVP, which resist oxidation as reductant and chelator respectively, were added to compare with the previous NaCl method (Cui et al. 2006) and isopropyl alcohol was used instead of alcohol to increase sedimentation. Several minor steps were modified in the improved NaCl method, such as 15 min centrifuge instead of 8 min for RNA pellet sedimentation. Total RNA was routinely obtained from 30 mg of fresh urediniospores using the improved methods, and gave a level of 3.3, 3.1 and 2.4-fold improvement than that (7.5 μg) obtained through liquid nitrogen grinding and using the Total RNA Isolation Kit from 30 mg urediniospores (Peng et al. 2007).
The three improved methods are different in their supe-riority for extracting different quantities of materials. In this study, the improved NaCl method was better for a large
quantity of material, while the SDS method was better for ex-tracting RNA from a small quantity of material. The improved LiCl method was similar to the results when extracting RNA from more than 15 mg, but not as good as the improved NaCl method when the material quantity was smaller than 15 mg. All three improved methods did not have material specificity, such as urediniospores vs. wheat leaves, and for leaves no matter inoculated or non-inoculated with P. striiformis f. sp. tritici. However, the improved NaCl method was proven to be the best in extracting RNA either from urediniospores or wheat leaves among the three methods.
The bands of PCR products detected by a pair of primers for elongation factor 1 alpha (EF1) of P. striiformis f. sp. tritici at the mid-sporulation and late-sporulation stages were clearly observed using agarose gel electrophoresis, although total RNA were degradated in these two stages. The RNA of P. striiformis f. sp. tritici within wheat leaves was just a small proportion of the total RNA extracted from the infected leaves, while the most of RNA comes from wheat leaf tissue which was nearly dead in late-sporulation stage of wheat stripe rust. The total RNA from both the pathogen and host tissue was degraded as observed in the agarose gel.
The molecular detection method was more quick and sensitive than visual detection, especially before sporula-tion. At the same time, the yields of total RNA obtained from the same quantity of healthy leaves and diseased leaves were different. The RNA quantity of healthy leaves was higher than diseased leaves using any improved methods. The integrity of RNA from the initial symptom-ex-pression stage was the highest among the four tested stages. However, the integrity of RNA from the mid- and late-sporulation stages was relative lower compared to other stages. Those might be because the pathogen and host were interacting to each other in these two stages, and the host had a hypersensitive response, which made host tissue and P. striiformis f. sp. tritici dead and resulted in degradation of the RNA. While the leaves during the initial symptom-expression stage were still fresh, intact RNA was observed in the agarose gel.
4. Conclusion
Comparison of the three improved methods (LiCl, SDS and NaCl), the NaCl method has a huge potential for application in large-scale studies with a huge number of field sam-ples. This method could be used to study overwintering of P. striiformis f. sp. tritici over a large region for predicting spring epidemics by detecting overwintered pathogen levels. Although the NaCl method was developed for extracting RNA from wheat leaves and urediniospores of P. striiformis f. sp. tritici, we think that this method can be used for studies on any other plants and pathogens.
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5. Materials and methods
5.1. Wheat planting and sample collecting
Seeds of wheat cultivar Mingxian 169, susceptible to stripe rust, were planted in 10 plastic pots (10 cm×10 cm×10 cm), and the seedlings were grown in a controlled growth chamber (16 h light/8 h dark at 14–16°C, illumination inten-sity 10 000 lux, relative humidity 60–80%) (Shang 2008). Seedlings inoculated with fresh urediniospores of Chinese P. striiformis f. sp. tritici race CYR 32 when the first leaves were expanded 10 days after planting (leaf development 11-BBCH scale). Using the shaking off method (Li and Shang 1989), fresh urediniospores were collected and mixed with talcum powder (1:20) in a test tube, and sealed with dou-ble-deck gauzes. Wheat leaves were dust-inoculated with the spore-talc mixture by tipping the tube. The inoculated seedlings were kept in a dew chamber at 8–10°C for 24 h and then put them in a growth chamber at 14–16°C and 16-h light/8-h dark cycles. Meanwhile, 10 pots of Mingxian 169 were also grown without inoculation as the control. Leaf samples were collected when initial symptoms (chlorotic patches without sporulation) appeared on inoculated leaves 9 dpi. At the same time, non-inoculated control samples were also collected.
In a field at Yangling, Shaanxi, Mingxian 169 was planted in 15 rows of 10 meters long and 30 cm between rows on October 6, 2011. The field was managed according to the local cultural practices. About two weeks after planting, fresh urediniospores of CYR 32 and talcum powder (1:20) were mixed and inoculated using the method described above onto the seedlings of five lines in the middle of the field which were sprayed with water in advance. A plastic film was used to cover the inoculated plants, and was removed in the next day. Leaf samples were collected at different time points after inoculation to represent different infection stages of wheat stripe rust covering the latent period, initial symptom-expression, mid-sporulation and late sporulation stages 5, 9, 20, and 25 dpi, respectively.
5.2. New method for completely transferring materi-als from mortars and tubes
Urediniospores of P. striiformis f. sp. tritici were weighted in a 1.5-mL centrifuge tube (non-sterile certified free of RNase and DNase, the Gold Standard Cat., Beijing, China). The tube was filled with 40 μL Tris-HCl buffer (pH 8.0) per 10 mg material, gently shaken, and kept at room temperature for 5 min in order to let Tris-HCl buffer permeate into ured-iniospores. The tube was flash-frozen in liquid nitrogen to make it into solid ice, quickly transferred the solid ice into
a mortar containing liquid nitrogen, and the spore solid ice were quickly ground. A volume of 20 μL Tris-HCl (pH 8.0) per 10 mg material was added into the mortar, ground twice. The powder was transferred to a clean 2.0-mL centrifuge tube for further use. Meanwhile, the same volume of RNA-free water was also used to evaluate its function in transferring materials instead of Tris-HCl (pH 8.0).
Wheat leaves were weighed (ca. 100.0 mg), flash-frozen, transferred into a mortar pre-cooled with liquid nitrogen, and ground quickly. A volume of 50 μL of Tris-HCl (pH 8.0) per 100 mg was added into the mortar, ground twice, and the powdery was transferred into a clean 2.0 μL centrifuge tube for further use.
5.3. Isolation and purification of total RNA using improved LiCl, SDS and NaCl methods
Typically, RNA was isolated from 100 mg of ground tissue powder prepared in liquid nitrogen, according to the in-structions of three reported and economic RNA extraction methods including LiCl (Yu et al. 2007), SDS (Yu et al. 2007), and NaCl (Cui et al. 2006). In this study, the three methods were improved. The improved methods are described as the following (Table 4): 1) 800 μL extraction buffer was used for the three improved methods, respectively. The mixes were vigorously vortexed twice for 90 s each after 10 μL 1% β-mercaptoethanol and 16% PVP were added, which resist oxidation as reductant and chelator, respectively. The tubes were incubated for 10 min on ice; 2) to each of solutions, 5 mol L–1 sodium acetate (pH 4.8) was added at the rate of one third and 750 μL of phenol:chloroform:isoamyl alcohol (25:24:1, pH>7.8, Solarbio, Cat. No. P1012) was added, shaken vigorously until the two phases form an emulsion, and incubated for 8 min on ice; 3) the mix was centrifuged at 12 000 r min–1 for 15 min at 4°C. The aqueous phase was collected and re-extracted with 700 μL chloroform and 1/3 volume of 5 mol L–1 sodium acetate (pH 4.8); 4) RNA was precipitated with 800 μL isopropyl alcohol and 1/10 volume of 3 mol L–1 sodium acetate (pH 5.2) at –20°C for 40 min; 5) the tube was centrifuged at 12 000 r min–1 for 15 min at 4°C, the supernatant was discarded, and RNA pellet was washed with 75% ethanol and absolute ethanol, air-dried and solved in 20 μL RNA-free water; 6) 5 μL 10× DNase I buffer, 5 U DNase I, 20 U RNase inhibitor, and RNA-free water up to 50 μL were added. The solution was incubated for 30 min at 37°C. 7) The total RNA in the solution was precipitated with 3 volume isopropyl alcohol and 1/10 vol-ume of 3 mol L–1 sodium acetate (pH 5.2) at –20°C for 40 min. 8) The tube was centrifuged at 12 000 r min–1 for 15 min at 4°C, and the pellet was washed with 75% ethanol and absolute ethanol, respectively. The RNA pellet was
1301MA Li-Jie et al. Journal of Integrative Agriculture 2016, 15(6): 1293–1303
Tabl
e 4
Com
paris
on o
f RN
A e
xtra
ctio
n m
etho
ds fr
om u
redi
nios
pore
s of
and
whe
at le
aves
infe
cted
by
P. s
triifo
rmis
f. s
p. tr
itici
Ste
psR
efer
ence
met
hod
Impr
oved
met
hod
LiC
l (Y
u et
al.
2007
)S
DS
(Yu
et a
l. 20
07)
NaC
l (C
ui e
t al.
2006
)Li
Cl
SD
SN
aCl
Mat
eria
l grin
ding
Grin
d in
liqu
id n
itrog
enG
rind
in li
quid
nitr
ogen
Grin
d in
liqu
id n
itrog
enA
dd 4
0 μL
Tris
-HC
l bu
ffer (
pH 8
.0) p
er 1
0 m
g m
ater
ials
; kee
p at
room
te
mpe
ratu
re fo
r 5 m
in,
flash
-froz
en a
nd g
rind
in
liqui
d ni
troge
n; a
dd 2
0 μL
Tris
-Hcl
(pH
8.0
) per
10
mg
ured
inio
spor
es o
r 50
μL
Tris
-Hcl
(pH
8.0
) pe
r 100
mg
leav
es tw
ice
durin
g gr
indi
ng
Add
40
μL T
ris-H
Cl
buffe
r (pH
8.0
) per
10
mg
mat
eria
ls; k
eep
at ro
om
tem
pera
ture
for 5
min
, fla
sh-fr
ozen
and
grin
d in
liq
uid
nitro
gen;
add
20
μL T
ris-H
cl (p
H 8
.0) p
er
10 m
g ur
edin
iosp
ores
or
50 μ
L Tr
is-H
cl (p
H 8
.0)
per 1
00 m
g le
aves
twic
e du
ring
grin
ding
Add
40
μL T
ris-H
Cl
buffe
r (pH
8.0
) per
10
mg
mat
eria
ls; k
eep
at ro
om
tem
pera
ture
for 5
min
, fla
sh-fr
ozen
and
grin
d in
liq
uid
nitro
gen;
add
20
μL T
ris-H
cl (p
H 8
.0) p
er
10 m
g ur
edin
iosp
ores
or
50 μ
L Tr
is-H
cl (p
H 8
.0)
per 1
00 m
g le
aves
twic
e du
ring
grin
ding
Ext
ract
ion
buffe
r1
mL
(100
mm
ol L
–1 L
iCl;
100
mm
ol L
–1 T
ris;
100
mm
ol L
–1 E
DTA
; 1%
SD
S);
1%
β-m
erca
ptoe
than
ol;
16%
PV
P
1 m
L (1
00 m
mol
L–1
LiC
l;
100
mm
ol L
–1 T
ris;
50 m
mol
L–1
ED
TA;
1% S
DS
);
1% β
-mer
capt
oeth
anol
800
μL (2
00 m
mol
L–1
N
aCl;
20 m
mol
L–1
Tris
-H
Cl (
pH 8
.0);
5 m
mol
L–1
E
DTA
; 1%
SD
S)
800
μL (1
00 m
mol
L–1
LiC
l; 10
0 m
mol
L–1
Tris
; 100
mm
ol L
–1
ED
TA; 1
% S
DS
);
1% β
-mer
capt
oeth
anol
; 16
% P
VP
800
μL (1
00 m
mol
L–1
LiC
l; 10
0 m
mol
L–1
Tris
; 50
mm
ol L
–1
ED
TA; 1
% S
DS
);
1% β
-mer
capt
oeth
anol
; 16
% P
VP
800
μL (2
00 m
mol
L–1
N
aCl;
20 m
mol
L–1
Tris
- H
Cl (
pH 8
.0);
5 m
mol
L–1
ED
TA; 1
% S
DS
);
1% β
-mer
capt
oeth
anol
; 16
% P
VP
Pol
ysac
char
ide
and
pigm
ent r
emov
al1/
3 vo
lum
e of
5 m
ol L
–1
NaA
c (p
H 4
.8)
1/3
volu
me
of 5
mol
L–1
N
aAc
(pH
5.2
)–
1/3
volu
me
of 5
mol
L–1
N
aAc
(pH
4.8
)1/
3 vo
lum
e of
5 m
ol L
–1
NaA
c (p
H 4
.8)
1/3
volu
me
of 5
mol
L–1
N
aAc
(pH
4.8
)
Pro
tein
rem
oval
Ext
ract
pro
tein
w
ith 2
× vo
lum
e of
ch
loro
form
:isoa
myl
al
coho
l (49
:1)
Ext
ract
pro
tein
2 ti
mes
w
ith a
n eq
ual v
olum
e of
ph
enol
:chl
orof
orm
:isoa
myl
al
coho
l (25
:24:
1)
800
μL
phen
ol:c
hlor
ofor
m:is
oam
yl
alco
hol (
25:2
4:1)
; 800
μL
chlo
rofo
rm
750
μL
phen
ol:c
hlor
ofor
m:is
oam
yl
alco
hol (
25:2
4:1)
; 700
μL
chl
orof
orm
and
1/3
vo
lum
e of
5 m
ol L
–1 N
aAc
(pH
4.8
)
750
μL
phen
ol:c
hlor
ofor
m:is
oam
yl
alco
hol (
25:2
4:1)
; 700
μL
chl
orof
orm
and
1/3
vo
lum
e of
5 m
ol L
–1 N
aAc
(pH
4.8
)
750
μL
phen
ol:c
hlor
ofor
m:is
oam
yl
alco
hol (
25:2
4:1)
; 700
μL
chlo
rofo
rm a
nd 1
/3 v
olum
e of
5 m
ol L
–1 N
aAc
(pH
4.8
)
RN
A p
reci
pita
tion
Pre
cipi
tate
with
1/1
0 vo
lum
e of
3 m
ol L
–1
NaA
c (p
H 5
.2) a
nd
an e
qual
vol
ume
of
isop
ropy
l alc
ohol
at –
20°C
ov
erni
ght,
and
prec
ipita
te
agai
n w
ith 6
00 μ
L bu
ffer
(2 m
ol L
–1 L
iCl;
50 m
mol
L–1
ED
TA)
Pre
cipi
tate
with
an
equa
l vo
lum
e of
isop
ropy
l al
coho
l at –
20°C
ove
rnig
ht
Pre
cipi
tate
with
1/1
0 vo
lum
e of
3 m
ol L
–1 N
aAc
(pH
5.2
) and
2.5
vol
ume
of
pre-
cool
ed a
bsol
ute
alco
hol
in –
70°C
for 1
5–30
min
800
μL is
opro
pyl a
lcoh
ol
and
1/10
vol
ume
of 3
mol
L–1
sod
ium
ace
tate
(pH
5.
2) a
t –20
°C fo
r 40
min
800
μL is
opro
pyl a
lcoh
ol
and
1/10
vol
ume
of 3
mol
L–1
sod
ium
ace
tate
(pH
5.
2) a
t –20
°C fo
r 40
min
800
μL is
opro
pyl a
lcoh
ol
and
1/10
vol
ume
of 3
mol
L–1
sod
ium
ace
tate
(pH
5.
2) a
t –20
°C fo
r 40
min
1302 MA Li-Jie et al. Journal of Integrative Agriculture 2016, 15(6): 1293–1303
air-dried and dissolved in 20 μL RNA-free water. 9) The RNA concentration was measured with a NanoDrop 2000 spectrophotometer (Thermal Co., USA).
5.4. Evaluation of RNA extraction
RNA samples were assessed by the quantity, quality and integrity. The OD260/230 absorbance ratio was used to indicate polysaccharide or polyphenol contamination, and the OD260/280 ratio was used to determine any protein contamination (Manning 1991). The values of OD260/230 ratio greater than 2.0 and the OD260/280 ratio between 1.8 and 2.0 were considered no polysaccharide/polyphenol and protein contaminations, respectively (Asie et al. 2000). RNA integrity was determined by visualizing in an agarose gel electrophoresis.
5.5. PCR reactions
The reverse transcriptase reaction of RNA was carried out at 37°C for 15 min using a Prime Script® RT Reagent Kit (TaKaRa Biotechnology Co., Dalian, China), followed by heat inactivating the avian myeloblastosis virus reverse transcriptase (AMVRT) at 85°C for 5 s. Elongation factor 1 of P. striiformis f. sp. tritici was used as specific primers to detect the pathogen (Yin et al. 2009). The size of target product was 159 bp. The PCR reactions were performed in a thermal cycler (Bio-Rad MyCycler, USA). Each reaction contained 0.5 U Taq, 2.0 μL buffer (Mg2+ free), 1.2 μL MgCl2
(25 mmol L–1), 1.6 μL dNTPs mixture (10 mmol L–1 each), 0.4 μL primer mix (containing 20 μmol L–1 of each primer), 1 μL (30 ng) of cDNA, and distilled water up to 20 μL. The amplification conditions were as the following: A denaturation step at 94°C for 1 min followed by 35 amplification cycles consisting of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min, with a final extension step at 72°C for 10 min. PCR products were run in a 2.5% agarose gel and visualized under UV light after staining in an ethidium bromide solution (0.5 μg mL–1) for 15 min.
5.6. Data analysis
Turkey test based on the one-way ANOVA were performed using Statistic Package for Social Science (SPSS) 16.0 version.
Acknowledgements
This work was supported by the National Key Basic Re-search Program of China (2013CB127700) and the Na-
tional Natural Science Foundation of China (31071640 and 31271985), and partially supported by the 111 Project from Education Ministry of China (B07049).
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(Managing editor ZHANG Juan)
