Elsevier

Agri Gene

Volume 7, March 2018, Pages 52-58
Agri Gene

Transgenic expression of a maize geranyl geranyl transferase gene sequence in maize callus increases resistance to ear rot pathogens

https://doi.org/10.1016/j.aggene.2018.01.001Get rights and content

Highlights

  • A geranyl geranyl transferase-like gene (GGT) was cloned from a maize ear rot resistant inbred.

  • Callus containing the GGT transgene was more resistant to maize pests than control GUS callus.

  • Headspace ethanol production was often increased in the callus with the GGT gene.

  • Higher levels of ethanol were associated with higher levels of fungal resistance.

Abstract

Determining the genes responsible for pest resistance in maize can allow breeders to develop varieties with lower losses and less contamination with undesirable toxins. A gene sequence coding for a geranyl geranyl transferase-like protein located in a fungal ear rot resistance quantitative trait locus was cloned from an inbred with reported resistance to Fusarium proliferatum and Fusarium verticillioides ear rot. Transgenic expression of the gene in maize callus reduced colonization by these two Fusarium species and also Fusarium graminearum relative to a β-glucuronidase (GUS) transformant control. Some transformants were also more insect resistant. The more fungal resistant transformant lines produced higher levels of headspace ethanol which were significantly associated with antifungal activity, especially for F. verticillioides. Maize pyruvate decarboxylase appears to have a moiety capable of interacting with the geranyl geranyl transferase, suggesting ethanol production is enhanced due to more efficient transfer of pyruvate through the mitochondrial membrane. Other undetermined mechanisms may also be enhancing resistance of the transformants to the Fusarium fungus, however. This is the first report of the involvement of a geranyl geranyl transferase-like sequence in fungal resistance in plants, and represents a novel mechanism for producing higher yielding and better quality maize.

Introduction

Economic losses due to maize insects and diseases are considerable worldwide (Oerke, 2006). Additionally ear rot pathogens reduce grain quality and can also contaminate the grain with toxins (mycotoxins) that are hazardous to humans and animals and are strictly regulated worldwide (Van Egmond and Jonker, 2005). Many different strategies have been explored to reduce the problems with maize pests. One of the most effective is the use of host plant resistance (Fritsche-Neto and Borem, 2012). Problems with maize foliar pathogens (Carson, 1999), stalk rots (White, 1999) and insect pests (Smith, 2005) have been greatly reduced through breeding efforts. Incorporation of bacterial genes into maize has greatly reduced damage due to insects, although many markets find transgenic materials objectionable (Gilbert, 2013), and several pests have developed resistance to the transgene product (Fatoretto et al., 2017). There is still a considerable need to identify genes involved in maize insect and disease resistance in order to promote incorporation by breeding or new technologies such as CRISPR/Cas.

Many studies on ear rot pathogens that are mycotoxin producers have identified regions of chromosomes, called quantitative trait loci (QTL) that are associated with resistance trait inheritance. A number of these studies were subjected to meta-analysis, and identified many chromosomal regions that are associated with inherited resistance through the comparison of progeny of crosses of resistant and susceptible inbreds (Xiang et al., 2010). However, these QTLs involve thousands of genes, requiring additional efforts to determine which genes are most relevant to pursue in breeding for increased resistance. QTL mapping, coupled with the sequencing of the maize genome, has made it possible to identify candidate genes potentially involved in insect and fungal resistance. A meta QTL associated with resistance to ear rot and mycotoxin producing fungi Aspergillus flavus, Fusarium proliferatum, F. verticillioides and F. graminearum spanned portions of chromosome 3 (Xiang et al., 2010).

As part of this process, we identified a geranyl geranyl transferase-like gene (previously annotated in Gramene as a terpene cyclase) located in a resistance QTL on chromosome 3. Geranyl geranyl transferases (GGTs) are responsible for adding a long chain hydrocarbon to a protein to assist in membrane interactions and protein-protein interactions (Running, 2014). In several instances, proteins derivatized by geranyl groups have been associated with plant pathogen resistance in different plant species (Crowell and Huizinga, 2009). Because of a potential role in plant disease resistance, we decided to investigate this role through transgenic expression in maize callus along with evaluation of potential increases in resistance to representative maize ear insect pests and mycotoxin-producing ear rot pathogens. The gene was cloned and expressed in maize callus, which was evaluated for resistance to different species of maize pest insects and ear rot fungi. The relationship between resistance seen and potential gene product influences was determined through chemical analysis.

Section snippets

Insects

Corn earworms (Helicoverpa zea) and fall armyworms (Spodoptera frugiperda) were reared on pinto bean based diet as described previously (Dowd, 1988). First instars were used for bioassays.

Fungi

Original stocks of F. graminearum (strain III-B, David Schisler, from Illinois), F. proliferatum (NRRL 13569, NRRL Culture Collection, originally from California) and F. verticillioides (AMRF-4, Robert Proctor, from Illinois) were collected from maize.

Transgenic callus production

The candidate gene was located as part of a selection

Gene analysis

The cloned gene sequence was 537 bp (Genbank MG676680), coding for a 178 amino acid protein of molecular weight 19,467, with a − 8.65 charge and a pI of 4.58. It was 18 amino acids more than the original consensus Gramene sequence of 160 amino acids, which diverged at amino acid 38 but was otherwise similar with the exception of the gap in that region. Many variants are reported for the sequence in Gramene, which when accessed 13Aug2017 listed the location as 16,786,916–16,799,630, with 9

GGT functional determinants and protein substrates

Although containing the prenyl transferase domain, the sequence of the gene we cloned seems inconsistent with prior reports of functional GGTs. However, similarly defined proteins are reported from C. elegans and mice. Maize genomic sequences reported in Gramene were highly variable, but some had the same start position. This information suggests that the gene is undergoing evolutionary selection and perhaps has adopted new functionality, the ability to derivatize enzymes associated with

Conclusions

Transgenic expression of a GGT-like coding sequence from a Fusarium resistant maize inbred conferred enhanced insect and disease resistance compared to GUS controls. The increased pathogen resistance was often significantly associated with the relative levels of ethanol in headspace volatiles. There were portions of two enzymes involved in ethanol production from pyruvate, pyruvate decarboxylase and alcohol dehydrogenase that could potentially interact with the GGT is a manner that would

Disclaimer

Mention of trade names or commercial products in the article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer.

Conflict of interest

The authors declare that they have no conflicts of interest.

Acknowledgements

We thank M. Doehring and D. Lee for technical assistance; the USDA, Agricultural Research Service, North Central Regional Plant Introduction Station for providing seed of maize inbred NC300; D. Schisler for providing the initial stock strain of F. graminearum; the NRRL Culture Collection for providing the initial stock strain of F. proliferatum, R Proctor for providing the initial stock strain of F verticillioides; and A.P. Rooney for comments on prior versions of the manuscript.

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