Resistance Evaluation to Sheath Blight in Transgenic Rice Lines

Transcription

1 Rice Science, 24, 11(3): Resistance Evaluation to Sheath Blight in Transgenic Rice Lines LI Ai-hong 1, 3, XU Xin-ping 2, DAI Zheng-yuan 3, CHEN Zong-xiang 1, LI Bao-jian 2, ZHANG Hong-xi 3, PAN Xue-biao 1 ( 1 Department of Agronomy, Agricultural College, Yangzhou University, Yangzhou 2259, China; 2 Bio-Engineering Center, Zhongshan University, Guangzhou 51275, China; 3 Lixiahe Agricultural Institute of Jiangsu Province, Yangzhou 2252, China) Abstract: Resistance of forty-one homozygous rice lines transformed with chitinase gene (RC24) and β-1,3 -glucanse gene (β-1,3-glu) to sheath blight was analyzed by inoculation. Among different lines, the resistance had significant differences according to the result by cluster analysis. The lines could be categorized into resistant, moderately resistant, moderately susceptible and susceptible types, while 92.1% of which belonging to moderately resistant or moderately susceptible type. For different resistant or susceptible lines, the resistance to rice sheath blight was remarkable correlated with the chitinase activity of transgenic lines except resistant type lines, in which enzyme activity coded by target gene was lower than moderately resistant type. The chitinase activity of transgenic lines tested at different time after inoculation or different organs of the same plant was uniform, which suggested that the expression of chitinase gene was constitutive in nature. Check varieties chitinase activity would change at different time after inoculation and reach a peak at sometime, but it had no difference at various parts of the same plant. Key words: rice; transgenic lines; sheath blight resistance; cluster analysis; chitinase activity Sheath blight (Rhizoctonia solani Kühn) is one of the three major rice diseases worldwide. The pathogen, R. solani is a semi-saprophytic fungus with a wide range of hosts [1, 2]. Because it is difficult to find ideal resistant germplasm for this disease, scientists tried to breed resistant cultivar by gene engineering approach. In previous studies, researchers had reported that rice materials harboring exogenous genes, such as chitinase gene, glucanse gene, etc., had good resistance to rice sheath blight [3-6]. For these results, there was an obvious shortcoming regarding the method of identification, in which the materials were evaluated at early segregating generation for resistance, or the results were obtained by investigating the degree of inhibiting fungus developing when it co-cultured with the liquid extracted from the leaves of transferred materials. This study was conducted by selecting transgenic homozygous lines as materials unlike previous most studies involving heterozygous materials, by improving inoculation and investigation method [7], creating identical condition, evaluating at appropriate time, to validate the reliability of introducing exogenous target gene to enhance the resistance to rice sheath blight, and to understand the distribution of resistance in the Received: 15 October 23; Accepted: 12 December 23 Corresponding author: PAN Xue-biao(panxb@pub.yz.jsinfo.net) generations transformed with target genes. At the same time, the expression mode of chitinase activity in the transgenic lines was also assayed. Materials MATERIALS AND METHODS Forty-one homozygous rice lines transformed with chitinase gene(rc24) and β -1,3-glucanse gene( β -1,3-Glu) from the rice variety Qisiruanzhan, were selected as the experimental materials, and the varieties of Qimiaoxiang and Lemont identified in the past studies as the resistant and susceptible check variety, respectively [8]. The methods of plasmid construction and molecular analyses were followed as in the reference [3]. Methods Measurement of resistance to rice sheath blight The experiment was conducted at the farm of the Agricultural College, Yangzhou University. Each material was planted with three rows including 45 plants with two replications. The key measures of managing were to provide moderate fertilizer and keep water continuously. RH-9, a R. solani strain with strong pathogenicity, provided by the Plant Protection Institute, Jiangsu Academy of Agricultural Sciences, was used for infection. Inoculation was carried out with short woody

2 96 Rice Science, 24, Vol. 11, No. 3, 24 toothpicks with a length of.8 1. cm. Autoclaved toothpicks were incubated with RH-9 strain on PDB medium for 3 days, then placed behind the leaf collar of the third sheath, counting from the top at the first stem elongation stage of growth. After inoculation, the status of the sheath-holding stem should be nearly unchanged. Resistance was measured about 3 days after heading using a 9 rating scale, as described by Rush et al [9]. Detection of chitinase activity Based upon the evaluation result of resistance to rice sheath blight, we selected resistant, moderately resistant, moderately susceptible, susceptible type lines, and detected the transgene expression activity. Because the target genes of RC24 andβ-1,3-glu were close linked in the plasmid, in this study, only chitinase activity was tested. The extraction of chitinase was done following the method as introduced by Hu et al [1]. Two grams of rice leaves were homogenized in liquid nitrogen with 1 ml HOAC-NaOAC (.1 mol/l, ph 5.6) and centrifuged at 15 g and 4 for 15 min. The supernatant was obtained for activity assay. The chitinase activity was tested with improved deoxidizing-sugar method [11], which was conducted with colloid chitin as substrate, and testing the OD value at the wavelength of 42 nm with K 3 [Fe(CN) 6 ], and drawing standard curve with N-acetaminoglucose. The enzyme quantity used to hydrolyze chitin colloid and generate 1 µmol/l deoxidizing-sugar was defined as one chitinase activity unit. Statistical analysis Normal variance and dynamic cluster analyses were made with the software SAS6.12. RESULTS The differences of resistance to sheath blight among transgenic lines Variance analysis The results of variance analysis indicated that the resistance of transgenic lines had significant differences (Table 1). The resistance rating of check varieties, including resistant variety Qimiaoxiang and susceptible variety Lemont, were similar to those results as reported by previous studies [2, 8], which also showed that the data obtained in this study were reliable. Cluster analysis Because the multiple comparison could not assort clearly the resistance rating, we attempted to classify the different transgenic lines with the method of dynamic cluster analysis. The results reflected that the transgenic lines could be divided into four groups according to their resistance level (Table 2): the first, resistant with disease rating of ; the second, moderately resistant ( ); the third, moderately susceptible ( ) and the fourth, susceptible type having a rating of An important information revealed in Table 2 was that there were 38 transgenic lines out of 41 with good or partial resistance to rice sheath blight, and only 3 lines similar to non-transgenic check variety appeared to susceptible reaction, which also revealed that the approach introduced target genes into ideal varieties could enhance the resistance to the pathogens. Of course, 92.1% were moderately resistant or moderately susceptible type in the 38 lines, and only 3 lines, F C, F C-1 and F C gained the resistant level. Whether their resistance were caused by exogenous gene expression, somaclonal variation, or insertional mutation, must be validated furthermore. Moreover, there were 3 lines, whose molecular analyses although were positive, developed susceptible symptoms like non-transgenic check variety, suggesting that it might be the result of gene silencing. Chitinase activity of transgenic lines Relationship between the resistance to rice sheath blight and the chitinase activity To affirm the expression of the introduced exogenous gene had an exertion on the resistance to rice sheath blight, we selected resistant, moderately resistant, moderately susceptible, and susceptible lines as materials, and non-transgenic variety Qisiruanzhan, susceptible variety Lemont, resistant variety Qimiaoxiang as check varieties, respectively, by assaying their chitinase activity, to study the relationship Table 1. Variance analysis on resistance to sheath blight among different rice lines. Sources of variance df SS MS F value P Among lines **.1 Among years Error Total

3 LI Ai-hong, et al. Resistance Evaluation to Sheath Blight in Transgenic Rice Lines 97 Table 2. Cluster analysis on resistance to sheath blight among different rice lines. Group No. Code of lines Resistance rating of 2 Resistance rating of 21 Average 1 4 F C F C Qimiaoxiang F C QI F C F A F C F C F C F A QI-3-2-D QI F C QI F F A F B F C F A F C F A F C F C F C F C F C F C F D F-18-1-C F D F C F C F D F B F D F C QI A QI F A Lemont F C QI Qisiruanzhan(CK) between the chitinase activity expression and the resistance to rice sheath blight. The major results obtained (Fig. 1) showed that: (i) There were endogenous chitinase both in susceptible and resistant varieties, and this chitinase activity was very low if no inducement from exterior circumstance. After being transformed with exogenous gene RC24, the susceptible variety Qisiruanzhan showed a high chitinase activity because of its over-expression. (ii) For different resistant or susceptible type lines, by detecting its chitinase

4 98 Rice Science, 24, Vol. 11, No. 3, 24 Chitinase activity (U/g FW) Chitinase activity (U/ g) R MR MS S CK-NT CK-S CK-R R MR MS S CK-NT CK-S CK-R Resistant or susceptible type Inoculation time (h) Fig. 1. Chitinase activity of different type transgenic lines. R, Resistant lines; MR, Moderately resistant lines; MS, Moderately susceptible lines; S, Susceptible lines; CK-NT, Non-transgenic check variety; CK-S, Susceptible check variety; CK-R, Resistant check variety. Fig. 2. Chitinase activity of transgenic lines at different time after inoculation. R, Resistant lines; MR, Moderately resistant lines; MS, Moderately susceptible lines; S, Susceptible lines; CK-NT, Non-transgenic check variety; CK-S, Susceptible check variety; CK-R, Resistant check variety. activity, we found that the rice sheath blight resistance was significantly correlated with the chitinase activity of transgenic lines except resistant type lines, and the enzymatic activity encoded by target gene in resistant type lines was 3 U lower than that in the moderately resistant type, which also suggested that there might be some other reasons to explain its high resistance. Dynamic variation of chitinase activity in transgenic lines Previous studies had reported that many physical or chemical factors, such as pathogens infection, salicylic acid, ethylene, phytohormone, mechanical stimulation, etc., could induce chitinase activity in plants [11-13]. By inoculating pathogen of R. solani on transgenic lines to induce the production of chitinase activity, and detecting chitinase activity of the inoculated leaves, we may find that: (i) For resistant, moderately resistant, moderately susceptible lines, their chitinase activity had little change and distributed with a parallel mode because of the constitutive expression of target genes. (ii) For non-transgenic regular varieties, whether susceptible varieties Qisiruanzhan and Lemont or resistant variety Qimiaoxiang, their chitinase activity appeared to be distributed with a flexuous mode. Susceptible varieties had a small increment in the chitinase activity after inoculation, but resistant variety had more higher. Furthermore, all varieties, including resistant and susceptible type, reached the peak in the chitianse activity of expression 72 h after inoculation. (iii) The resistance ratings of susceptible transgenic lines were similar to non-transgenic parent, which also supported that it might be the result of gene silencing. Seventy-two hours after inoculation, the chitinase activity tested at different inoculation locations of the Chitinase activity (U/g) Flag leaf The 2nd top leaf Sh eat h R MR MS S Transgenic lines CK-NT CK-S CK-R Check variety Fig. 3. Chitinase activity of different parts of the same rice plant for transgenic lines. R, Resistant lines; MR, Moderately resistant lines; MS, Moderately susceptible lines; S, Susceptible lines; CK-NT, Non-transgenic check variety; CK-S, Susceptible check variety; CK-R, Resistant check variety.

5 LI Ai-hong, et al. Resistance Evaluation to Sheath Blight in Transgenic Rice Lines 99 same plant, such as the 1st top leaf, the 2nd top leaf and sheath etc. had no distinct differences, regardless of that it was in transgenic lines or resistant and susceptible regular varieties. The similarity of chitinase activity in transgenic lines may be the result of constitutive expression of target gene, as for regular varieties, it might suggest the inductive signal can transduct with a high speed. DISCUSSION For a long time, rice resistance breeding to sheath blight hasn t been developed fleetly because of its infection character of pathogen and absence of ideal resistant germplasm [2, 14, 15]. Many researchers reported that they had obtained varieties or germplasm with good resistance to fungi pathogen by transferring different chitinase genes [16], but in which the resistance assay was conducted only at initial stages or laboratory stages, and couldn t give an objective evaluation to high generation of population. In this study, we found when susceptible variety Qisiruanzhan was transformed with exogenous RC24 and β-1,3-glu gene, its resistance to rice sheath blight had significant differences among lines from different transformants. By cluster analyzing, the transgenic lines could be divided into resistant, moderately resistant, moderately susceptible, susceptible types, but 92.1% of which belonged to the type of moderately resistance or moderately susceptible. Further more, we detected the enzyme activity of target gene, and observed that its expression in moderately resistant type lines was the strongest one, and the resistant type lines were 3 U lower than moderately resistant type in enzymatic activity of target gene. Recently, we also found these three resistant lines had good resistance to different physiological races of rice blast fungus, and their basic agronomic traits had been greatly changed, such as the delay of heading date, enhancement of plant height and 1-grain weight, etc. But in the transgenic lines whose agronomic traits hadn t been modified, the resistant type couldn t be identified. Earlier research had ever reported that resistant rice germplasm to sheath blight could be obtained by protoplast culturing [17], so, we speculated that their good resistance might have relationship to abundant somaclonal variation or inserting mutation of target gene that could activate the defense system in plants, and the approach of transferring exogenous RC24 and β-1,3-glu gene might only partly enhance the resistance to sheath blight and couldn t make it reach the resistant level. In this study, we also observed the existence of endogenous chitinase in resistant or susceptible type varieties and this activity was very low if no any inducement from foreign circumstance. By inducing of inoculation, the susceptible varieties had a small enhancement in the chitinase activity, but the resistant type had a big one and reached a high level. The receptor parent Qisiruanzhan, susceptible to sheath blight and rice blast, when it was transformed with exogenous RC24 and β-1,3-glu gene and induced by inoculation, the constitutive expression of target gene could inhibit or cover up the effect made by endogenous chitinase gene and made its distribution with a parallel way. These raised many questions. Such as, the plasmid used to transform in this study contained an alkalecence chitinase gene from IR36, whether the inhibiting effect was generated because the endogenous and exogenous chitinase genes were all from rice? If using another chitinase gene that was not coloned from rice, is there it? For a rice variety with high-inducing effect in endogenous chitinase activity, when it is transformed with exogenous target gene, is there in existence of inhibiting effect to restrict its enhancement of resistance? All of these questions are worth to further study. 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