Necesidad de la formación de valores en escolares con

are annotated with an identifiable promoter region upstream of the TSS. Addition-

ally, previous studies have shown that regulatory regions may lie within the introns

(Schauer et al., 2009) or the 3’ UTR (Cawley et al., 2004). The findings presented

here have been derived from the upstream promoter sequences. The method used

can equally be applied to any orthologous or paralogous sequences that are thought

to contain functionally conserved regulatory regions, and doing so may uncover more

functional CNSs.

The high rate of “binding site turnover” means that regulatory elements mu-

tate rapidly over evolutionary time but maintain their functional role, albeit having

little sequence conservation, as demonstrated in

Drosophila

(Moses et al., 2006).

Therefore, the approach presented here could not account for the full complement

of conserved regulatory elements within plant genomes. However, the loss-free na-

ture of the method means that it is able to find all alignment conserved sequences,

when provided with the appropriate set of orthologous sequences for comparison.

Alternative methods, such as the alignment-free model developed by Koohy et al.

(2010) can be used in order to find elements that are functionally, but not alignment,

conserved. Methods that combine comparative genomics with other resources, such

as gene expression data (as in (Vandepoele et al., 2006; Heyndrickx and Vandepoele,

2012; Spangler et al., 2012)) are also useful in aiding the discovery and analysis of

regulatory modules.

2.5

Conclusions

In this chapter, highly conserved noncoding sequences (CNSs) identified using a com-

parative genomic approach, were predicted to be involved in the transcriptional reg-

ulation of their associated genes. In addition, it was found that the CNS-associated

genes themselves commonly had a role in transcriptional regulation. The finding

that regulatory genes are themselves highly regulated makes biological sense; as

plants rely on their regulatory machinery to integrate signals from internal and ex-

ternal stimuli to formulate a complex response, it is intuitive to put those genes

under strict control. Taking into account the CNS length and binding site content,

the prediction can be made that each gene is likely to have a number of regulators

that can interact directly with the promoter DNA, and others that potentially op-

erate indirectly through protein protein interactions with DNA-bound regulators.

Furthermore, several thousand binding sites have been predicted to be mediating

TF-gene links in the gene regulatory network ofA. thaliana. The implication of this

finding is that the strongly maintained CNSs and the genes they are associated with

play an intrinsic role in the regulatory network that is shared among dicot plants.

Chapter 3

Elucidating Functional Elements

and Gene Regulatory Network

Using Yeast One-Hybrid

Screens

3.1

Introduction

Over the next few decades the world population is predicted to grow by another 2.5

billion, to reach 9.5 billion. Sustaining a growing population means increasing crop

yields in a sustainable fashion. One of the challenges associated with increased crop

yields is increasing resistance of plants to a variety of biotic and abiotic stresses. Past

research links increased stress resilience with decreased growth and yield (Herms and

Mattson, 1992). Therefore understanding plant responses to stresses means that a

better balance can be attained in the future crop with the help of genetic engineer-

ing. The necrotrophic fungusBotrytis cinerea

accounts for an estimated 15-40% of

harvest losses in grape varieties and 20-25% in strawberry crops and has a broad

host range, infecting more than 200 plant species also including tomato (Elad et al.,

2007a). Studies on the model organism Arabidopsis have identified a number of

genes that play a major role in resistance to Botrytis, including PDF1.2 (Penninckx

et al., 1996; Zimmerli et al., 2001), BOS1 (Mengiste et al., 2003) and PAD3 (Fer-

rari et al., 2007). However, many more genes and mechanisms that are involved in

increased resistance to the fungus are yet to be characterised. Additionally, mech-

anisms of gene regulation are not well understood and simply increasing expression

of defence resistant genes leads to adverse effects on the overall growth phenotype of

the plant (Clarke et al., 2001; Hua et al., 2001; Jambunathan et al., 2001). Similarly,

gain-of-function double mutants restore growth but increase the susceptibility of the

plant to a variety of infections (Shirano et al., 2002; Zhang et al., 2003). Therefore

a better understanding of the regulation of gene expression would allow for fine

tuning of the expression of disease resistant genes whilst minimising the negative

effects such as reduced growth.

In the previous chapter putative regulatory regions along the promoter DNA

in Arabidopsis were identified using the APPLES software package. The promoter

DNA sequences of Arabidopsis were compared to the promoter sequences of a va-

riety of other, closely and distantly related, plant species. Hundreds of CNSs were

identified with varying degrees of conservation across multiple species and multiple

lines of evidence point at the functional nature of the CNSs. The aim of this chap-

ter is, firstly, to identify a small set of genes that are regulated by the same TF(s)

using time-series mRNA expression profiles associated with the response to the in-

fection with Botrytis. The promoters of the identified genes will be interrogated

further for information determining their regulation using Yeast One-Hybrid (Y1H)

library screens. The library screen allows a picture of the gene regulatory network

(GRN) to be built from the bottom up, focusing on all possible protein-DNA inter-

actions associated with particular promoters. TF(s) regulating selected genes may

also serve as master regulators in the stress related network against Botrytis as a

whole and therefore would be good targets for further experimentation. This knowl-

edge would contribute to our understanding of the regulation of Botrytis defence

responses by identifying direct protein-DNA interactions for differentially expressed

genes regulated in the infection process.

3.1.1

Available Experimental Techniques For Probing Protein-DNA