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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
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