Efficacy of Hydroponics 110

Though conceptually hydroponic co-cultivation more closely represents conditions in

planta, more work is required in uncovering the roles these detectable genes have in plant defense and their potential application as candidate targets to corroborate the suggested

superior utility of hydroponics to study Agrobacteria-Arabidopsis interaction. However

by comparing the transcriptomic profiles of shoot and root tissues to previously detected

A. thaliana transcriptomic changes in response to A. tumefaciens using conventional models, the overlap in detectable transcripts is suggestive of the power of using

hydroponic co-cultivation. When comparing the responses of root tissues, there is greater overlap with transcriptomic responses detected using site-specific wounding based

inoculation techniques (Appendix 3 and 7). This suggests that the absence of typical plant morphology in plant cell suspension cultures creates greater dissimilarity in detectable host responses in comparison to systems that maintain whole plant structure. The lack of greater similarity between hydroponics and site-specific wounding based techniques is likely attributed to mechanically wounding the host that ultimately influences the

detectable A. thaliana Col-0 transcriptomic output. By evading any artificial introduction

of direct contact between A. thaliana Col-0 and A. tumefaciens C58 post-inoculation into

liquid culture, the detection of A. tumefaciens C58 virulence is imperative to ensure

23), not only are host responses testable using hydroponics, but A. tumefaciens C58 responses to host perception can also be analyzed. Over a fifty-six hour period, the

growth of A. tumefaciens C58 in the hydroponic co-cultivation system was monitored

every 4 hours. Interestingly, A. tumefaciens growth was stagnant leading to the twenty-

hour period where growth sharply declined followed by a sudden spike in growth (Figure

24). The delay in A. tumefaciens growth responses indicates an initial lag period, however

following the spike, growth oscillates up and down with a gradual increase until the final time point. Though atypical of growth curves, this finding suggests some back and forth

communication is occurring due to the presence of A. thaliana Col-0. This interesting

finding requires more attention in order to determine how this seemingly unique interaction is being established.

4.3.1

Alternative Applications of Co-cultivation in Hydroponics

In addition to the benefit hydroponic co-cultivation offers in the study of A. thaliana Col-

0 and A. tumefaciens C58 interaction, there are a variety of broader applications as well.

The versatility of hydroponics for studying plant-stimuli responses extends beyond the

Arabidopsis-Agrobacterium interaction presented here. Hydroponic co-cultivation can be applied to elucidate plant responses to a wide variety of additional stimuli including other pathogenic or beneficial microbes and chemical signals, as well as enabling the study of microbial responses that closely mimic rhizosphere conditions by avoiding the disruption of natural root secretions. This produces a “real-time” stimulation of microbial virulence, which is more progressive than the sudden artificial induction of virulence resulting from acetosyringone (AS) supplementation. Since the secretion and concentration of these compounds in liquid is time sensitive, time-course studies can be conducted to monitor temporal chemical alterations to the liquid solution and the variations in dynamic plant- microbe interactive responses. In addition to stimuli interchangeability, a variety of plant hosts can be utilized for response analyses (Figure 4). Yet, there are a variety of

parameters to consider when cultivating host models, namely mechanical and biological challenges. Identifying plant models that maintain typical plant morphology is of

significant importance. Discrepancies in typical root development may impair the host’s ability to indirectly communicate with microbes, weakening any possible subsequent direct interaction. In addition, dependent on seed and root size, adjustment in the use of metal mesh and growth container may also be required to ensure root structures fully develop below the mesh surface and shoot tissues proliferate above (Figure 4).

Hydroponics under lab conditions offers additional flexibility in applied pathogens and/or chemical compounds. In the case of bacterial, fungal and herbivory pathogens, the sole requirement is that the liquid media and host can support their survival to facilitate infection/feeding. Insects acting above the root surface can be applied directly to various non-submerged regions of the system (i.e.: mesh platform, plant structure, sides of the hydroponic tank, etc.). Moreover, hydroponics may offer new insight into studying the effects of pre-existing (allelochemicals) and/or applied (synthetic) chemical compounds by introduction into the liquid medium, as well as effects of variation in environmental conditions, as has been previously attempted using other methodologies including root dipping [67,68]. The plasticity in stimuli that can be studied using this system makes hydroponics beneficial for identifying molecular plant responses to a variety of abiotic- biotic stimuli.

4.3.1.1

Gene expression pattern analysis

Developing an innovative system for plant-pathogen interaction creates possibility to detect molecular processes that have not been previously identified. This is not limited to interactive studies between host-microbe, but may also reveal other host properties such as spatial/temporal expression patterns of genes and their associative annotations. During the process of reviewing gene annotations for differentially expressed transcripts detected in root and shoot tissues, a small subset of genes identified had not been

previously detected in their corresponding tissue sites previously. From systemically affected tissue sites, three DEGs had not been previously detected in shoot tissues (Appendix 29). On the other hand, 58 DEGs were detected in our root transcriptome that

have not been previously identified in root tissues (Appendix 30). The relatively smaller amount of DEGs uniquely identified to be expressed in shoot tissue using the hydroponic co-cultivation system is the result of analyzing entirely indirectly affected tissue (leaf, stem, shoot). If the expression patterns were analyzed independently for different indirectly affected sites then it is more likely that many more genes that have not been detected in those specified tissues may have been identified for expression. Together these unintentional findings suggest that by further developing more accurate models for experimental study, we are able to better mimic and uncover host-microbe interactions

occurring in planta.