Expression Profiling of Four Defense-Related Buffalograss

Expression Profiling of Four Defense-Related Buffalograss Transcripts in Response to Chinch Bug (Hemiptera: Blissidae) Feeding Author(s): Crystal Ramm , Aaron Saathoff , Teresa Donze , Tiffany Heng-Moss , Frederick Baxendale , Paul Twigg , Lisa Baird , and Keenan Amundsen Source: Journal of Economic Entomology, 106(6):2568-2576. 2013. Published By: Entomological Society of America URL: http://www.bioone.org/doi/full/10.1603/EC13267

BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder.

BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research. PLANT RESISTANCE Expression Proﬁling of Four Defense-Related Buffalograss Transcripts in Response to Chinch Bug (Hemiptera: Blissidae) Feeding

CRYSTAL RAMM,1 AARON SAATHOFF,2 TERESA DONZE,1 TIFFANY HENG-MOSS,1,3 1 4 5 6 FREDERICK BAXENDALE, PAUL TWIGG, LISA BAIRD, AND KEENAN AMUNDSEN

J. Econ. Entomol. 106(6): 2568Ð2576 (2013); DOI: http://dx.doi.org/10.1603/EC13267 ABSTRACT Oxidative enzymes are one of many key players in plant tolerance responses and defense signaling pathways. This study evaluated gene expression of four buffalograss transcripts (two peroxidases, a catalase, and a GRAS (gibberellic acid insensitive [GAI], repressor of GAI, and scarecrow) and total peroxidase activity in response to western chinch bug (Blissus occiduus Barber) feeding in susceptible and resistant buffalograsses (Buchloe¨ dactyloides (Nuttall) Engelmann). Basal levels of all four transcripts were consistently higher in the resistant buffalograss when compared with the susceptible genotype, which suggests important physiological differences exist between the two buffalograsses. The four defense-related transcripts also showed differential expression between infested and control plants for both the resistant and susceptible buffalograsses. Differences in total peroxidase activity were also detected between control and infested plants, and basal peroxidase activity was higher in the resistant genotype. Overall, this study indicates that elevated basal levels of speciÞc peroxidases, catalases, and GRAS may be an effective buffalograss defense strategy against chinch bug feeding and other similar biotic stresses.

KEY WORDS Buchloe¨ dactyloides, Blissus occiduus, plant resistance, peroxidase, catalase

A nationwide emphasis on water and energy con- The potential for identifying chinch bug-resistant servation as well as environmental and groundwater buffalograsses was Þrst suggested by differences in safety concerns have encouraged development of susceptibility of several buffalograsses to the western turfgrass cultivars requiring less water, fertilizer, mow- chinch bug (HengÐMoss et al. 2002). Of the 200 ge- ing, and pesticide usage than conventional varieties. notypes evaluated for chinch bug resistance, the ge- Over the last two decades, buffalograss (Buchloe¨ dac- notype Ô378Õ was identiÞed as the most susceptible and tyloides (Nuttall) Engelmann) has emerged as a prom- ÔPrestige,Õ the most resistant, was categorized as tol- ising turfgrass species because of its low maintenance erant (HengÐMoss et al. 2002, 2003; Gulsen et al. 2004). requirements and relative freedom from arthropod HengÐMoss et al. (2004) reported increased levels of pests and disease (Pozarnsky 1983, Wu and Harivandi peroxidase activity following chinch bug feeding in 1989, Riordan 1991, Riordan et al. 1996). In the 1990s, Prestige and a loss of catalase activity in 378. Gulsen et the western chinch bug, Blissus occiduus Barber, al. (2010) also found increased levels of peroxidase emerged as a serious pest of buffalograss (Baxendale activity in Prestige in response to chinch bug feeding et al. 1999). Feeding by this insect causes serious and suggested that the elevated levels of peroxidases injury that can ultimately result in death of the plant in the tolerant buffalograsses allowed the plant to (Baxendale et al. 1999). Buffalograss resistance to detoxify peroxides and other reactive oxygen species chinch bugs, when used as part of an integrated pest (ROS) that accumulate as a result of plant stress. management (IPM) program, has the potential to ef- Several studies (Felton et al. 1994a,b; Constabel et al. fectively and economically reduce chinch bug infes- 2000; Hiraga et al. 2000; Chaman et al. 2001) have tations, while minimizing pesticide inputs and main- documented similar increases in oxidative enzymes tenance efforts. (lipoxygenase, polyphenol oxidase, superoxide dismu- tase, catalase, and peroxidase) in response to arthro- 1 Department of Entomology, 105 Entomology Hall, University of pod feeding, while other studies suggest that increases Nebraska, Lincoln, NE 68583. in these important defense-related enzymes may con- 2 Grain, Forage, and Bioenergy Research Unit, USDAÐARS, Plant tribute to plantÕs insect resistance (Hildebrand et al. Science Hall 362F, Lincoln, NE 68583. 3 Corresponding author, e-mail: [email protected]. 1986, Van der Westhuizen et al. 1998, Ni et al. 2001, 4 Department of Biology, 335 Bruner Hall, University of Nebraska, HengÐMoss et al. 2004, Rangasamy et al. 2009, Gulsen Kearney, NE 68449. et al. 2010, Pierson et al. 2011). As a Þrst step toward 5 Department of Biology, 5998 Alcala Park, University of San Diego, understanding the role oxidative enzymes and the San Diego, CA 92110. 6 Department of Agronomy and Horticulture, Plant Science Hall speciÞc mechanisms and genes contributing to toler- 377H, University of Nebraska, Lincoln, NE 68583. ance responses in buffalograss, subtractive cDNA li- December 2013 RAMM ET AL.: BUFFALOGRASS TRANSCRIPTS IN RESPONSE TO CHINCH BUG 2569

braries and Illumina sequencing databases were de- collected with an aspirator. Chinch bugs were held in veloped using control and chinch bug-infested 378 the laboratory for 24 h, and injured and dead chinch (susceptible) and Prestige (resistant) buffalograsses bugs were discarded before initiating the experiment. (P.T., unpublished data, Wachholtz et al. 2013). Tran- The treatment design was arranged asa2by2by3 scripts found to be differentially expressed between factorial with two buffalograss genotypes (Prestige the resistant and susceptible buffalograss genotypes and 378), two chinch bug infestation levels (control were identiÞed and categorized according to molec- and infested), and three sampling dates (5, 12, and 19 d ular function. Four defense-related transcripts includ- after chinch bug introduction). Ten chinch bugs ing two peroxidases (POX and POX-1), a catalase (fourth and Þfth instar) were introduced onto the leaf (CAT), and a GRAS (gibberellic acid insensitive blades of each designated infested plant. Chinch bugs [GAI], repressor of GAI, and scarecrow) were se- were conÞned on the plants using tubular Plexiglas lected for further investigation. The peroxidase and cages (4 cm in diameter by 30 cm in height) covered catalase transcripts were selected based on earlier with organdy fabric at the top and fastened by rubber work that suggested their involvement in the response bands. Control plants were caged in a similar manner. of buffalograss to chinch bug infestation (HengÐMoss After chinch bug introduction, plants were main- et al. 2004, Gulsen et al. 2010). The GRAS transcript tained in the greenhouse until each respective sam- was selected because this family of plant-speciÞc tran- pling date. The experimental design was a completely scription factors has been shown to be upregulated in randomized design with six replications. response to pathogens and other biotic stresses (May- At each evaluation date, chinch bug numbers were rose et al. 2006). recorded and plants were rated for chinch bug damage The overall focus of this study was to evaluate gene following previously established procedures (HengÐ expression of four defense-related transcripts and to- Moss et al. 2002). Plant samples from the crown, leaf tal peroxidase activity in response to chinch bug feed- blades, and lower leaf sheaths were collected for RNA ing in susceptible and resistant buffalograsses. Infor- extraction, immediately frozen in liquid nitrogen, and mation on the role of these defense-related transcripts stored at Ϫ80ЊC. Total RNA was extracted from the and the speciÞc mechanisms and genes contributing to frozen plant tissue from a minimum of three biological the resistance will enhance our understanding of the replications using TRIzol reagent following manufac- physiological mechanisms associated with buffa- turer protocol (Invitrogen, Carlsbad, CA), followed lograss tolerance to chinch bugs. In addition, under- by puriÞcation using the RNeasy MinElute Cleanup standing these mechanisms at a molecular level may Kit and associated protocol (Qiagen, Valencia, CA). facilitate the identiÞcation of phenotypic character- RNA was visualized using gel electrophoresis. istics and development of markers that would pro- TaqMan assays were performed using a 7500 Fast foundly impact the breeding of turfgrasses with en- Real-Time PCR System following the manufacturerÕs hanced tolerance to chinch bugs. protocol (7500 Fast Real-Time PCR System, Applied Biosystems, Foster City, CA). Primers and probes for the catalase (CAT), peroxidase (POX), and GRAS Materials and Methods transcripts were designed using Primer Express soft- Study 1: Gene Expression of Three Plant Defense- ware (Applied Biosystems; Supp. Tables 1Ð3 [online Related Transcripts. Sod plugs (10.6 cm in diameter by only]). A control assay was developed for ubiquitin- 8 cm in depth) of the resistant buffalograss Prestige conjugating enzyme (UCE; Czechowski et al. 2005, and susceptible buffalograss 378 were collected from Gutsche et al. 2009). Each plate was loaded in tripli- the John Seaton Anderson Turfgrass and Ornamental cates for each sample. Each 20 ␮l reaction contained Research Facility (JSA Research Facility), University 10 ␮lof2ϫ Master Mix, 0.5 ␮lof40ϫ Multiscribe and of Nebraska Agricultural Research and Development RNase Inhibitor Mix, 4 ␮l of RNA (diluted to 10 ng/ Center, near Mead, NE. These two buffalograss cul- ␮l), and 1 ␮l1ϫ TaqMan probe mix. This mix con- tivars are known to differ in chinch bug resistance, tained forward and reverse primers and probe DNA ␮ ploidy level, and other turfgrass performance traits. and 4.5 lH2O. The PCR reaction cycling conditions Individual stolons of each genotype were planted in were 48ЊC for 30 min, 95ЊC for 10 min, 95ЊC for 15 s, “SC-10 Super Cell” single cell 3.8-cm-diameter by 21- and 60ЊC for 1 min for 40 cycles. Analysis of cycle cm-deep cone-tainers (Stuewe & Sons, Inc., Corvallis, threshold data were completed using the ⌬⌬Ct OR). The soil mixture was a ratio of 2:1:3:3 sand: soil: method with the UCE as the endogenous control (Li- peat: perlite. Buffalograss plants were watered and vak and Schmittgen 2001). fertilized (20N-10P-20K soluble) as needed. Plants Damage ratings were analyzed using a mixed model were maintained at a temperature of 24 Ϯ 3ЊC and a (PROC MIXED, SAS Institute 2002) to detect differ- photoperiod of 16:8 (L:D) h under 400-watt high- ences between the two buffalograsses. Means were intensity discharge lamps. separated using Fisher least signiÞcant difference Chinch bugs were collected from buffalograss re- (LSD) procedure when appropriate (P Յ 0.05). For search plots at the JSA Research Facility by vacuum- qRT-PCR, raw data were entered into LINREG (Ra- ing the soil surface with a modiÞed ECHO Shred NЈ makers et al. 2003) to estimate average reaction efÞ- Vac (model #2400, ECHO Incorporated, Lake Zurich, ciency that was used to generate efÞciency-corrected IL). Chinch bugs were sifted through a 2-mm mesh Ct values. The efÞciency-corrected Ct values were screen and fourth and Þfth instar chinch bugs were then used to generate ⌬Ct and ⌬⌬Ct values (Livak and 2570 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 106, no. 6

Schmittgen 2001). Relative expression differences tions. Four biological replicates of each treatment were calculated using 2Ϫ⌬⌬Ct. Statistical signiÞcance were analyzed in triplicate. of the ⌬⌬Ct values was determined using the Wil- Enzymatic activity for peroxidases was measured coxon rank-sum test available through the SAS PROC using a spectrophotometer. To assess peroxidase ac- NPAR1WAY procedure (SAS Institute 2002). Type I tivity, 5 ␮l buffalograss protein extract was diluted error was controlled at ␣ ϭ 0.05. Changes in transcript with 20 ␮l 20 mM HEPES buffer, pH 6.0. Each well was level were determined for each control and infested loaded with 5 ␮l diluted extract in triplicate (three buffalograss genotype (e.g., infested Prestige vs. con- assays per sample ϭ 15 ␮l). Peroxidase activity was trol Prestige) as well as between Prestige and 378 analyzed using a modiÞed protocol from Hildebrand control plants at each time point. et al. (1986) and HengÐMoss et al. (2004). The activity Study 2: Gene Expression and Kinetics of Two Per- was determined by monitoring the increase in absor- oxidases. To further evaluate the role of peroxidases in bance at 470 nm for 2 min. Each sample reaction the defense response of buffalograss to chinch bug (triplicate) was initiated by adding 75 ␮lof18mM feeding, a second study was conducted to evaluate guaiacol, 25 ␮l of 3% hydrogen peroxide, 20 ␮lof200 gene expression of POX and POX-1 (Supp. Table 4 mM HEPES buffer pH 6.0, and 75 ␮l of distilled water [online only]), and total peroxidase activity. Buffa- to microplate wells containing 5 ␮l of plant extract. lograss plants were established following procedures Peroxidase speciÞc activity was calculated using the similar to those described in Study 1. The treatment molar absorptivity of guaiacol at 470 nm (26.6 by 103). design was arranged asa2by2by3factorial with two Resulting data for total protein content and perox- buffalograss genotypes (Prestige and 378), two chinch idase activity were analyzed using SAS Version 9.2 bug infestation levels (control and infested), and Mixed model analysis (SAS Institute 2002, Cary, NC) three sampling dates (5, 12, and 19 d after chinch bug and PROC GLIMMIX procedure to reveal differences introduction). Twenty chinch bugs (third, fourth, Þfth in total protein content and peroxidase activity be- instar, and adults) were introduced onto the leaf tween genotypes and treatments. blades of each designated infested plant. Chinch bugs were again conÞned on the plants using tubular Plexi- Results and Discussion glas cages (4 cm in diameter by 30 cm in height) with organdy fabric fastened by rubber bands at the top. Study 1: Gene Expression of Three Defense-Related Control plants were also caged to ensure similar en- Transcripts. Chinch Bug Damage. Overall, Prestige had vironmental conditions. After chinch bug introduc- lower damage ratings than 378 in response to chinch tion, plants were maintained in the greenhouse until bug feeding. Mean damage ratings (ϮSEM) for Pres- each respective sampling date. The experimental de- tige were 1.0 Ϯ 0, 1.4 Ϯ 0.24, and 2.2 Ϯ 0.20 for days sign was a completely randomized design with eight 5, 12, and 19, respectively. Mean damage ratings replications. Transcript abundance for POX and (ϮSEM) for 378 were 1.6 Ϯ 0.24, 2.75 Ϯ 0.25, and 3 Ϯ POX-1 was evaluated using the procedures described 0 for days 5, 12, and 19, respectively. These results are in Study 1 for tissue preparation and qRT-PCR anal- consistent with the Þndings reported by HengÐMoss ysis. Primers and probes for the POX-1 transcript were et al. (2002, 2003) and Gulsen et al. (2004, 2010). designed using Primer Express software (Applied Bio- Basal Transcript Levels of Noninfested (Control) systems). Four biological replicates of each treatment Plants. At 5 and 12 d, control plants of Prestige had were analyzed. higher CAT and GRAS transcript abundance levels Previously frozen plant tissue was also prepared for than control plants of 378 (CAT: D5: P Ͻ 0.0001; D12: soluble protein extraction using the Minute Total Pro- P Ͻ 0.0001; GRAS: D5: P Ͻ 0.0001; D12: P ϭ 0.01; Fig. tein Extraction Kit for Plant Tissues (Invent Biotech- 1). However, by day 19, no differences in CAT or nologies, Inc., Eden Prairie, MN) following the man- GRAS transcript abundance were detected between ufacturer protocol but without the denaturing lysis control plants of 378 and Prestige (CAT: P ϭ 0.59; buffer, and with the following modiÞcations: after GRAS: P ϭ 0.41; Fig. 1). Prestige control plants con- grinding 0.06 g tissue in liquid nitrogen with mortar sistently had higher levels (1.5- to 5-fold) of POX and pestle, tissue was homogenized as per the spin- transcript abundance than 378 control plants at all column protocol with addition of 2 ␮l plant cocktail times (D5: P Ͻ 0.0001; D12: P Յ 0.0001; D19: P ϭ 0.02; inhibitor (modiÞed protocol from HengÐMoss et al. Fig. 1). 2004) and 100 ␮l native buffer (from Minute kit). Transcript Levels of Infested Plants. Prestige showed Tubes were centrifuged at 10,000 ϫ g for 10 min at 4ЊC. higher CAT transcript expression in control plants at Homogenate was collected and prepared for protein 5d(P ϭ 0.05) and similar expression levels at 12 d and and peroxidase analysis using a modiÞed protocol from by 19 d. Infested plants had higher CAT transcript Hildebrand et al. (1986) and HengÐMoss et al. (2004). abundance than their respective controls (D12: P ϭ Total protein content was measured by diluting 2 ␮l 0.27; D19: P Ͻ 0.0001; Fig. 2a). Infested 378 plants of buffalograss extract with 60 ␮l of water and using 10 consistently had higher CAT abundance than control ␮l of this dilution per assay for commercially available plants at 5 and 19 d (D5: P Ͻ 0.0001; D19: P ϭ 0.02), bicinchoninic acid (BCA) protein assay (Pierce, but no differences were detected between control and Rockford, IL). Bovine serum albumin was used as the infested plants at 12 d (P ϭ 0.10; Fig. 2a). Changes in standard for protein concentration. Procedures were POX transcript abundance were observed in Prestige carried out according to PierceÕs protein assay instruc- in response to chinch bug feeding (Fig. 2b). Prestige December 2013 RAMM ET AL.: BUFFALOGRASS TRANSCRIPTS IN RESPONSE TO CHINCH BUG 2571

8 D5 7 D12 D19

6 * 5 *

4 * *

Fold ChangeFold 3

2 * * *

0 CAT GRAS POX Transcript Fig. 1. Transcript abundance between resistant Prestige and susceptible 378 control plants at days 5, 12, and 19 for each designated transcript. A fold change Ͼ1 represents higher transcript abundance in control plants of Prestige. A fold change Ͻ1 indicates higher transcript abundance in control plants of 378. A fold change equal to 1 indicates no difference between control plants of either genotype. (*) indicates a signiÞcant difference (P Յ 0.05). (Online Þgure in color.) showed differences between control and infested similar levels of transcript abundance (D19: P ϭ 0.76; plants at 5 and 12 d with higher levels of POX tran- Fig. 2b). Increased transcript abundance of POX was script expression in control plants at both time points observed in susceptible 378 in response to chinch bug (D5: P Ͻ 0.0001; D12: P ϭ 0.01), but by day 19, had feeding at 5 d and 12 d with no differences detected

8 8 D5 D5 7 a D12 7 b D12 D19 D19

6 6

5 5

4 4

3 3

2 2 * * * * * 1 * 1 * * 0 0 Prestige 378' Prestige 378'

8 D5 7 c D12 D19

6 Fold Change 5

2 1 * 0 Prestige 378' Genotype Fig. 2. CAT (a), POX (b), and GRAS (c) transcript abundance between control and infested Prestige and 378 plants at days 5, 12, and 19. A fold change Ͼ1 represents higher transcript abundance in infested plants while a fold change of Ͻ1 indicates higher transcript abundance in control plants of the indicated genotype. A fold change equal to 1 indicates no difference in transcript abundance between control and infested plants. (*) indicates a signiÞcant difference (P Յ 0.05). (Online Þgure in color.) 2572 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 106, no. 6

8 * D5 7 D12 D19 6 *

Fold ChangeFold 3 * * 2 * 1 *

0 POX POX-1 Transcript Fig. 3. POX and POX-1 transcript abundance between susceptible 378 and resistant Prestige control plants at days 5, 12, and 19. A fold change Ͼ1 represents higher transcript abundance in control plants of Prestige. A fold change Ͻ1 indicates higher transcript abundance in control plants of 378. A fold change equal to 1 indicates no difference between control plants of either genotype. (*) indicates a signiÞcant difference (P Յ 0.05). (Online Þgure in color.) between control and infested plants at 19 d (D5: P Ͻ Study 2. Gene Expression and Kinetics of Two Per- 0.0001; D12: P ϭ 0.01; D19: P ϭ 0.21; Fig. 2b). Prestige oxidases. Chinch Bug Damage. Overall, Prestige had plants showed higher GRAS transcript abundance in lower damage ratings than 378 in response to chinch control plants at5d(P ϭ 0.002; Fig. 2c). No differ- bug feeding. Mean damage ratings (ϮSEM) for Pres- ences in GRAS transcript expression were detected tige were 1.0 Ϯ 0, 1.1 Ϯ 0.05, and 1.3 Ϯ 0.08 for days between control and infested plants at 12 and 19 d 5, 12, and 19, respectively. Mean damage ratings (D12: P ϭ 0.07; D19: P ϭ 0.83; Fig. 2c). The susceptible (ϮSEM) for 378 were 1.0 Ϯ 0, 1.6 Ϯ 0.22, and 2.3 Ϯ 0.28 genotype 378 showed no signiÞcant differences in for days 5, 12, and 19, respectively. These results are GRAS transcript abundance between control and in- consistent with Study 1. fested plants at any time (D5: P ϭ 0.14; D12: P ϭ 0.55; Basal Transcript Levels of NonInfested (Control) D19: P ϭ 0.15; Fig. 2c). Plants. Prestige control plants had higher POX tran- It is noteworthy that basal levels of POX in Prestige script abundance than 378 control plants at all three Ͻ Ͻ Ͻ control plants were higher than in 378 control plants time points (D5: P 0.0001; D12: P 0.0001; D19: P at all three time points (Fig. 1). These Þndings suggest 0.02; Fig. 3). For POX-1, Prestige control plants had that although infested Prestige plants initially have higher transcript abundance than 378 control plants at days 5 and 12, but 378 had greater transcript abun- lower levels of the POX transcript compared with dance at 19 d (D5: P Ͻ 0.0001; D12: P Ͻ 0.0001; D19: their respective control plants, they have higher basal P Ͻ 0.04; Fig. 3). levels of the POX transcript than the susceptible 378 Transcript Levels of Infested Plants. No differences and are therefore physiologically better prepared to in POX transcript abundance were detected between tolerate insect feeding. Prestige followed a similar Prestige control and infested plants at days 5 and 19 response for CAT and GRAS at 5 and 12 d, suggesting (D5: P ϭ 0.14; D19: P ϭ 0.10); however, control plants a potential role(s) for these two genes in the defense had higher POX transcript abundance than infested response of Prestige to chinch bugs. plants at 12 d (P ϭ 0.03; Fig. 4a). In response to chinch Buffalograss 378 plants showed differences between bug feeding, susceptible 378 showed higher POX tran- control and infested plants at 5 and 12 d with higher script abundance than control plants at all three time levels of POX transcript abundance in infested plants points (D5: P Ͻ 0.0001; D12: P Ͻ 0.0001; D19: P ϭ 0.01; at both time points (Fig. 2b). Further, 378 infested Fig. 4a). For POX-1, Prestige control plants showed plants had higher transcript abundance of CAT in higher transcript abundance at days 5 and 12; how- infested plants when compared with control plants at ever, by day 19 infested Prestige plants showed a 5 and 19 d (Fig. 2a). This increase in CAT and POX sevenfold higher POX-1 transcript expression than transcript abundance in infested 378 plants likely re- control plants (D5: P ϭ 0.03; D12: P Ͻ 0.0001; D19: P Ͻ sulted because the susceptible buffalograss had lower 0.0001; Fig. 4b). Susceptible 378 plants again showed basal levels of these two oxidative enzymes compared higher POX-1 transcript expression than control with the higher levels observed in Prestige. plants at all three times in response to chinch bug December 2013 RAMM ET AL.: BUFFALOGRASS TRANSCRIPTS IN RESPONSE TO CHINCH BUG 2573

8 D5 7 a D12 D19

5 *

4 * 3 * 2

1 *

0 Prestige 378'

8 * D5 7 b D12

Fold Change D19 6 * 5

4 * 3 * 2 1 * * 0 Prestige 378' Genotype

Fig. 4. POX (a) and POX-1 (b) transcript abundance between control and infested plants of Prestige and 378 at days 5, 12, and 19. A fold change Ͼ1 represents higher transcript abundance in infested plants while a fold change of Ͻ1 indicates higher transcript abundance in control plants of indicated genotype. A fold change equal to 1 indicates no difference in transcript abundance between control and infested plants. (*) indicates a signiÞcant difference (P Յ 0.05). (Online Þgure in color.) feeding (D5: P Ͻ 0.0001; D12: P Ͻ 0.0001; D19: P Ͻ Peroxidase activity. Total peroxidase activity be- 0.0001; Fig. 4b). It is important to note the differences tween control Prestige and 378 plants was greater in in the basal levels of these two transcripts between Prestige at 5 (t ϭ 3.97; df ϭ 14; P ϭ 0.001) and 12 d (t ϭ Prestige and 378 plants (Fig. 3). Higher POX and 4.81; df ϭ 14; P Ͻ 0.0001), but was similar between the POX-1 transcript abundance in the 378 plantÕs re- genotypes at 19 d (t ϭ 0.92; df ϭ 14; P ϭ 0.37; Fig. 5a). sponse to chinch bug feeding may be the result of Similar levels of peroxidase activity were also detected lower basal levels of these transcripts before chinch between control and infested Prestige plants at day 5 bug introduction. (t ϭϪ1.05; df ϭ 30; P ϭ 0.30; Fig. 5b). Peroxidase Total Protein. No differences in protein content activity was greater in Prestige plants in response to were detected between control Prestige and 378 chinch bug feeding at 12 d (t ϭϪ2.71; df ϭ 30; P ϭ 0.01; plants, or between control and infested Prestige and Fig. 5b) and 19 d (t ϭϪ2.02; df ϭ 30; P ϭ 0.05; Fig. 5b). 378 plants for any of the three time points (F ϭ 0.33; For the susceptible 378, no differences in activity df ϭ 2, 36; P ϭ 0.72). between control and infested plants were detected at 2574 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 106, no. 6

10 Prestige Control a 378 Control

8 * 6 *

mol/min*mg) 0 51219

10 10 Prestige Control 378 Control b c 378 Infested * Prestige Infested 8 8 *

6 6 *

Specific Activity Activity Specific (µ *

4 4

2 2

0 0 51219 51219 Day Fig. 5. Peroxidase speciÞc activity (micromoles per minute ϫ milligrams of protein) of control plants of buffalograsses Prestige and 378 (a) and of control and infested buffalograsses Prestige (b) and 378 (c). (*) indicates a signiÞcant difference (P Յ 0.05). day5(t ϭ 0.05; df ϭ 30; P ϭ 0.96; Fig. 5c). By 12 d, zymes, such as peroxidases and catalases, to help break infested plants had higher levels of total peroxidase down ROS (Laloi et al. 2004). In addition to their role ϭϪ ϭ ϭ activity than control plants (t 2.83; df 30; P in reducing H2O2 to oxygen and water, plant peroxi- 0.01), but at 19 d peroxidase activity was higher in dases have also been shown to play a role in cell wall control plants (t ϭ 2.39; df ϭ 30; P ϭ 0.02; Fig. 5c). synthesis, auxin catabolism, wound healing, oxidation These results support earlier Þndings by HengÐMoss of toxic reductants, and defense against pathogen and et al. (2004) and Gulsen et al. (2010). insect attack (Hiraga et al. 2001, Ni et al. 2001, Kawano In conclusion, this research documented the basal 2003, HengÐMoss et al. 2004). Of interest, the buffa- levels of four defense-related transcripts (CAT, POX, lograss POX transcript used in this study has sequence POX-1, and GRAS) and the abundance of these tran- identity with a peroxidase gene family expressed in scripts following chinch bug feeding on two buffa- rice in response to a bacterial pathogen (Chittoor et lograss genotypes, resistant Prestige and susceptible al. 1997). This further supports the idea that this tran- 378. In addition, total peroxidase activity was docu- script may play a role in the buffalograss defense mented before and in response to chinch bug feeding. response. Based on these studies, it is evident that Prestige and This study suggests the chinch bug-resistant geno- 378 are physiologically different with respect to their type Prestige is physiologically better prepared for basal levels of these four transcripts and differ in total biotic stresses than the susceptible genotype 378 based peroxidase activity. on the basal levels of the four defense-related tran-

ROS such as H2O2, superoxides, and hydroxyl rad- scripts CAT, POX, POX-1, and GRAS. Prestige may be icals are produced in response to insect and pathogen better able to handle much higher levels of ROS with- invasion. They are well known for their role as im- out cell death by maintaining elevated basal levels of portant early signaling molecules and for altering gene POX and CAT (Fig. 6). In contrast, lower basal levels expression patterns (Apel and Hirt 2004, Kotchoni and of these transcripts require chinch bug-susceptible Gachomo 2006, Pitzschke et al. 2006). Despite the 378 plants to expend energy in transcription and po- beneÞts gained from these defense signals, accumu- tentially translation of POX and CAT with the onset of lation of ROS can become toxic to plant cells (Laloi et insect feeding. They cannot, however, sustain the con- al. 2004). To protect themselves from the effects of tinued increases in transcript levels needed as insect ROS accumulation, plants have evolved scavenger en- feeding activity continues to increase over time. December 2013 RAMM ET AL.: BUFFALOGRASS TRANSCRIPTS IN RESPONSE TO CHINCH BUG 2575

ROS - screen other buffalograsses for the presence and up- SCAV - regulation of these genes, and may also provide valu- able markers for identifying resistant genotypes. Stress These Þndings also provide evidence that the resistant buffalograss Prestige may be better able to tolerate Stressss ssignalsals Defense chinch bug feeding by maintaining elevated basal lev- response els of the speciÞc transcripts CAT, POX, POX-1, and Metabolismolism GRAS. Furthermore, this work may lead to a better understanding of the defense mechanisms plants de- StressStress ssignalignal ploy to compensate for insect feeding. transductionransduction ROS production

ROS scavengerss References Cited Almagro, L., L. V. Go´mez Ros, S. Belchi–Navarro, R. Bru, A. Ros Barcelo´, and M. A. Pedren˜ o. 2009. Class III peroxidases in plant defense reactions. J. Exp. Bot. 60: 377Ð390. Fig. 6. A revised working hypothesis of the mechanism Apel, K., and H. Hirt. 2004. Reactive oxygen species: me- of buffalograss resistance to chinch bug feeding Þrst pro- tabolism, oxidative stress, and signal transduction. Annu. posed by HengÐMoss et al. (2004) and Gulsen et al. (2010). Rev. Plant Biol. 55: 373Ð399. Stress caused by insect feeding signals for the production of Baxendale, F. P., T. M. Heng–Moss, and T. P. Riordan. 1999. ROS as well as signaling transduction for defense-related Blissus occiduus Barber (Hemiptera: Lygaeidae): a new transcripts (DRT) such as CAT, POX, POX-1, and GRAS. chinch bug pest of buffalograss turf. J. Econ. Entomol. 92: High basal levels of DRT may allow the plant to both readily 1172Ð1176. detoxify increasing ROS, signal for increased ROS produc- Chaman, M. E., L. J. Corcuera, G. E. Zuniga, L. Cardemil, tion, and also signal for activation of genes involved in the and V. H. Argandona. 2001. Induction of soluble and cell buffalograss defense response in response to chinch bug wall peroxidases by aphid infestation in barley. J. Agric. pressure. Over time with increasing stress from chinch bug Food Chem. 49: 2249Ð2253. feeding, ROS may, in addition, behave as a signaling molecule Chittoor, J. M., J. E. Leach, and F. F. White. 1997. Differ- for increased expression of these DRT (speciÞcally ROS ential induction of a peroxidase gene family during in- scavengers) allowing the plant to better tolerate high levels fection of rice by Xanthomonas oryzae pv. oryzae. Mol. of ROS by actively breaking them down to nontoxic levels. Plant Microbe Interact. 10: 861Ð871. Constabel, C. P., Y. Peter, P. Lynn, J. Joseph, and M. E. Christopher. 2000. Polyphenol oxidase from hybrid Enhanced gene expression of the GRAS transcript poplar: cloning and expression in response to wounding has been documented in response to pathogen infec- and herbivory. Plant Physiol. 124: 285Ð296. tion and to the presence of gibberellins, plant hor- Czechowski, T. M., S. T. Altmann, M. K. Udvardi, and W. R. mones that have been implicated in the control of Scheible. 2005. Genome-wide identiÞcation and testing processes such as seed development, germination, and of superior reference genes for transcript normalization ßowering time (Mayrose et al. 2006). Mayrose et al. in Arabidopsis. Plant Physiol. 139: 5Ð17. (2006) also found the absence of a speciÞc GRAS gene Felton, G. W., C. B. Summers, and A. J. Mueller. 1994a. Oxidative responses in soybean foliage to herbivory by was a key factor in the susceptibility of tomato to bean leaf beetle and three-corned alfalfa leafhopper. infection by Pseudomonas syringae. Vandenabeele et J. Chem. Ecol. 20: 639Ð650. al. (2003) demonstrated that treatment of tobacco Felton, G. W., C. B. Summers, A. J. Mueller, and S. S. Duffey. plants with H2O2 resulted in higher level of a speciÞc 1994b. Potential role of lipoxygenases in defense against GRAS transcript, suggesting that GRAS may be an- insect herbivory. J. Chem. Ecol. 20: 651Ð666. other important defense-related transcript triggered Gulsen, O., T. M. Heng–Moss, R. Shearman, P. S. Baenziger, D. Lee, and F. P. Baxendale. 2004. Buffalograss germ- by H2O2. Although GRAS transcript expression was similar between control and infested plants of each plasm resistance to Blissus occiduus (Hemiptera: Lygaei- genotype over time, differences were detected at day dae). J. Econ. Entomol. 96: 2101Ð2105. Gulsen, O., T. Eickhoff, T. M. Heng–Moss, R. Shearman, F. 5 with Prestige control plants showing higher tran- Baxendale, G. Sarath, and D. Lee. 2010. Characteriza- script expression than their infested counterparts tion of peroxidase changes in resistant and susceptible (Fig. 2c). Higher basal levels of the GRAS transcript warm-season turfgrasses challenged by Blissus occiduus. in Prestige compared with 378 suggest that GRAS may Arthropod Plant Interact. 4: 45Ð55. be playing a role in the plantÕs resistance response to Gutsche, A., T. Heng–Moss, G. Sarath, P. Twigg, Y. Xia, G. Lu, chinch bug feeding. This is one of the Þrst studies to and D. Mornhinweg. 2009. Gene expression proÞling of document differential expression of a speciÞc GRAS tolerant barley in response to Diuraphis noxia plant transcript in buffalograss. (Hemiptera: Aphididae) feeding. Bull. Entomol. Res. 99: In summary, this research highlights the importance 163Ð173. Heng–Moss, T. M., F. P. Baxendale, T. P. Riordan, and J. E. of investigating basal levels of defense-related tran- Foster. 2002. Evaluation of buffalograss germplasm for scripts along with changes in gene expression in re- resistance to Blissus occiduus turf. J. Econ. Entomol. 95: sponse to insect feeding. The transcriptional proÞling 1054Ð1058. of the CAT, POX, POX-1, and GRAS defense-related Heng–Moss, T. M., F. P. Baxendale, T. P. Riordan, L. J. Young, transcripts provides a baseline that can be used to and K. Lee. 2003. Chinch bug-resistant buffalograss: and 2576 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 106, no. 6

investigation of tolerance, antixenosis and antibiosis. J. reproductive stage soybean to soybean aphid (Aphis gly- Econ. Entomol. 96: 1942Ð1951. cines Matsumura) feeding. Arthropod Plant Interact. 5: Heng–Moss, T. M., G. Sarath, F. P. Baxendale, D. Novak, S. 49Ð58. Bose, X. Ni, and S. Quisenberry. 2004. Characterization Pitzschke, A., C. Forzani, and H. Hirt. 2006. Reactive oxy- of oxidative enzyme changes in buffalograsses challenged gen species signaling in plants. Antioxid. Redox Sign. 8: by Blissus occiduus. J. Econ. Entomol. 97: 1086Ð1095. 1757Ð1764. Hildebrand, D. F., J. G. Rodriguez, G. C. Brown, K. T. Luu, Pozarnsky, T. 1983. Buffalograss: home on the range, but and C. S. Volden. 1986. Peroxidative responses of leaves also a turf grass. Rangelands 5: 214Ð216. in two soybean genotypes injured by twospotted spider Ramakers, C., J. M. Ruijter, R. H. Deprez, and A. F. Moor- mites (Acari: Tetranychidae). J. Econ. Entomol. 79: 1459Ð man. 2003. Assumption free analysis of quantitative real- 1465. time polymerase chain reaction (PCR) data. Neurosci. Hiraga, S., K. K. Yamamoto, H. Ito, K. Sasaki, H. Matsui, M. Lett. 13: 62Ð66. Honna, Y. Nagamura, T. Sasaki, and Y. Ohashi. 2000. Rangasamy, M., B. Rathinasabapathi, H. J. McAuslane, R. H. Diverse expression proÞles of 21 rice peroxidase genes. Cherry, and R. T. Nagata. 2009. Oxidative responses of FEBS Lett. 471: 245Ð250. St. Augustine grasses to feeding of southern chinch bug, Hiraga, S., K. Sasaki, H. Ito, Y. Ohashi, and H. Matsui. 2001. Blissus insularis Barber. J. Chem. Ecol. 35: 796Ð805. A large family of class III peroxidases. Plant Cell Physiol. Riordan, T. P. 1991. Buffalograss. Grounds Maintenance. 42: 462Ð468. February, pp. 12Ð14. Kotchoni, S. O., and E. W. Gachomo. 2006. The reactive Riordan, T. P., F. P. Baxendale, R. E. Gaussoin, and J. E. oxygen species network pathways: an essential prereq- Watkins. 1996. Buffalograss: an alternative native grass uisite for perception of pathogen attack and acquired for turf. Cooperative Extension, University of Nebraska, disease resistance in plants. J. Biosci. 31: 389Ð404. Lincoln, NE. Kawano, T. 2003. Roles of the reactive oxygen species-gen- SAS Institute. 2002. PROC userÕs manual, version 9.2. SAS erating peroxidase reactions in plant defense and growth Institute, Cary, NC. induction. Plant Cell Rep. 21: 829Ð837. Van der Westhuizen, A. J., X. M. Qian, and A. M. Botha. 1998. Laloi, C., K. Apel, and A. Danon. 2004. Reactive oxygen Differential induction of apoplastic peroxidase and chiti- signaling: the latest news. Curr. Opin. Plant. Biol. 7: 323Ð nase activities in susceptible and resistant wheat cultivars 328. by Russian wheat aphid infestation. Plant Cell Rep. 18: Livak, K. J., and T. D. Schmittgen. 2001. Analysis of relative 132Ð137. gene expression data using real-time quantitative PCR Vandenabeele, S., K. Van Der Kelen, J. Dat, I. Gadjev, T. and the 2(-Delta Delta C(T)) Method. Methods 25: 402Ð Boonefaes, S. Morsa, P. Rottiers, L. Slooten, M. Van Mon- 408. tagu, M. Zabeau, et al. 2003. A comprehensive analysis of Mayrose, M., S. K. Ekengren, S. Melech–Bonﬁl, G. B. Martin, hydrogen peroxide-induced gene expression in tobacco. and G. Sessa. 2006. A novel link between tomato GRAS Proc. Natl. Acad. Sci. U.S.A. 100: 16113Ð16118. genes, plant disease resistance and mechanical stress re- Wachholtz, M., T. Heng-Moss, P. Twigg, G. Lu, and K. sponse. Mol. Plant Pathol. 7: 593Ð604. Amundsen. 2013. Transcriptome analysis of two buffa- Ni, X., S. S. Quisenberry, T. M. Heng–Moss, J. Markwell, G. lograss cultivars. BMC Genomics (in press). Sarath, R. Klucas, and F. Baxendale. 2001. Oxidative re- Wu, L., and A. Harivandi. 1989. Buffalograss: promising, sponses of resistant and susceptible cereal leaves to symp- drought-resistant and here now. Golf Course Manag. 57: tomatic and nonsymptomatic cereal aphid (Hemiptera: 42Ð54. Aphididae) feeding. J. Econ. Entomol. 94: 743Ð751. Pierson, L. M., T. M. Heng–Moss, T. E. Hunt, and J. Reese. 2011. Physiological responses of resistant and susceptible Received 4 June 2013; accepted 23 August 2013.