Three candidate genes were selected from the transcriptomic changes detected in shoot tissues to potentially uncover use as targets to proactively improve host fitness and decrease susceptibility.
4.2.1
RLP32
Receptor-like proteins (RLPs), implicated primarily in defense and plant growth
regulation, are functionally and structurally similar to receptor-like kinases (RLKs)207.
RLPs associated with plant growth are under relatively high selective pressure in comparison to those associated with defense, which posses significant sequence
divergence among various plant genera208. A NCBI sequence BLAST209 of RLP32
revealed significant sequence divergence from that of alternative plant genus’. Based on
sequence divergence, a knockout of RLP32 may result in a loss of inducible plant defense
regulation causing constitutive expression of defense related genes directly linked to
RLP32 regulation. However, A. tumefaciens C58 showed little observational differences
in root attachment (Figure 21) on RLP32 knockdown plants. Despite detectable
differences in secretion profile by LC-MS analysis (Figure 22), the compounds
differentially detected may not influence chemotaxis of A. tumefaicnes C58 to root
structures. RLP32knockout strains did not have any unique detectable molecular features
and among those molecular features that were unique to all mutant strains in comparison
to the WT strain, RLP32 mutants had the most moderate profiles in terms of alterations to
compound detection (Figure 22). Although the elimination of RLP32 did not manifest in
increased A. thaliana Col-0 susceptibility to A. tumefaciens C58 irreversible root
attachment, it is possible that RLP32 functions solely within the host cytoplasm to
regulate the expression of defense proteins. Consequently, RLP32 would not have any
influence on secondary metabolite production; a factor likely required in regulating plant-
some direct role in intracellular defenses, it would be worth determining if A. thaliana
rpl32 -/- lines have observational differences in crown gal development.
4.2.2
PR-6 Proteinase Inhibitor
Pathogenesis-related (PR) proteins are inducible by effector-triggered immunity (ETI) and are effective antimicrobial, plant priming, plant structure re-enforcing and virulence
inactivating proteins210-211. Interestingly, although PR-6 family proteins are proteinase
inhibitors that antagonize plant infection by pathogenic fungi212, they are also strongly
induced in response to A. tumefaciens C58 eight hpi (Appendix 1) and the elimination of
PR-6 expression reduced A. tumefaciens C58 attachment (Figure 21). Perhaps the
comparably strong induction of a PR-6 proteinase inhibitor may not have been the result of activated defenses specifically targeting pathogenic bacteria, but instead was induced as a result of pathway crosstalk in systemically affected tissues. The activation of PR-6 proteins in systemic tissues by pathogen challenge suggests a preventative measure to decrease susceptibility of aerial regions of the host in the event of secondary
infection/infestation. It is unclear why irreversible root attachment of A. tumefaciens C58
was reduced, but this result seems to suggest alterations in host susceptibility to pathogen virulence. Though there were detectable differences in secretome profile in PR6
knockouts, it is unclear how the elimination of a PR protein would influence secondary metabolite biosynthesis and secretion. Instead, elimination of a PR6 protein inhibitor is likely to have an effect on influencing intracellular susceptibility. Though based on LC- MS analysis, it is clear that those molecular features unique to all mutant lines in
comparison to WT showed the greatest enhancement in PR6 protein knockouts (Figure 22). The reduction in irreversible attachment may be due to the increased production of
compounds that are antagonistic to pathogen challenge. Identifying the compounds differentially secreted in PR6 mutants may offer deeper insight to explain reduced pathogen attachment (Figure 22).
4.2.3
TEM1
Tempranillo (TEM) gene expression is directly correlated with the expression of genes
regulating flowering and biosynthesis of GA213. Overexpression of TEM genes results in
plants resembling GA-deficient mutants, and conversely, down-regulation results in
increases in GA content213. GA is a plant growth-promoting hormone that plays
important roles in diverse aspects of plant growth and development, such as stem
elongation214. In addition, GA has been shown to crosstalk with JA, mostly documented
by the antagonistic affects of GA on JA. Where GA is activated, JA mediated processes
are down regulated215. By analysis of TEM1 mutants, GA levels may be increased and
subsequently influence JA mediated defenses that have variable roles in plant defense, including production of secondary metabolites for secretion from the root structures. It is possible that there is reduction in root secretable compounds, regulated by JA, possessing antimicrobial properties, and the reduction in secretion is accounting for increased levels
of irreversible A. tumefaciens C58 root attachment (Figure 21). Based on LC-MS
analysis, there were a variety of molecular features that showed reductions in detection in
comparison to RLP32 and PR6 protein mutant strains upon pathogen challenge (Figure
22). The reduction in the production of these particular compounds may explain why
there was such a high level of irreversible A. tumefaciens C58 root attachment. The
enhanced production of these same compounds and the lower levels of A. tumefaciens
mutants is due to lower levels of production of these compounds (Figure 22).
Determining the compounds differentially produced and secreted in TEM1 knockouts
may offer deeper insight into secreted metabolites that may be interesting to study for
affects on A. tumefaciens C58 chemotaxis (Figure 22).