Regulation of plant defense responses downstream of PAMP receptors

The primary layer of plant immunity is pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). In PTI, when pattern recognition receptors (PRRs) recognize PAMPs, a highly regulated, rapid downstream signaling response such as activation of mitogen-activated protein (MAP) kinases, production of ROS and defense gene expression is initiated. My study involves the identification of MAP kinases activated in PTI and regulation of PTI responses by E3 ubiquitin ligases. In Arabidopsis thaliana, MPK3, MPK4, and MPK6 are activated upon PAMP treatment. However, previous studies suggest that there are more MAP kinases activated upon PAMP treatment. To identify the additional MAP kinases phosphorylated upon PAMP perception, transgenic plants expressing candidate MPKs with a ZZ-FLAG double tag (approx. 17kD) were generated in Col-0 background. Western blot analysis identified three MAP kinases, MPK1, MPK11 and MPK13 that are phosphorylated upon PAMP treatment. To identify E3 ligases involved in PTI, E3 ligases whose transcripts are up-regulated upon PAMP treatment were selected for our study. Transgenic lines overexpressing candidate E3 ligases were assayed for deficiencies in PTI. Overexpression of U6, one of the selected E3 ligases, lead to severe reduction of flg22-induced reactive oxygen species (ROS) production and increased susceptibility to Pseudomonas syringae pv. tomato (P.s.t.) DC3000 hrcC-. Furthermore, when U6 was overexpressed in BIK1 (a positive regulator of PTI)-HA transgenic plants, there was a decrease in BIK1-HA protein expression, leading to the hypothesis that BIK1 may be a potential target of U6. Overall, my thesis contributes to a better understanding of the signaling and negative regulation of PTI. Advancing our knowledge in plant immunity leads to the potential of its use in agriculture and plant protection.Science, Faculty ofBotany, Department ofGraduat

Regulation of MAP kinase cascades in plant immunity

In eukaryotes, MAP kinase cascades are important players of signal transduction in many biological processes. In plant immunity, two main MAP kinase pathways, MKK4/MKK5-MPK3/MPK6 and MEKK1-MKK1/MKK2-MPK4, are activated upon elicitor treatments. On the other hand, the YDA-MKK4/MKK5-MPK3/MPK6 cascade is involved in the negative regulation of stomatal differentiation. Plants have evolved different mechanisms to regulate the activity of these cascades. For example, brassinosteroid (BR) signaling has been reported to promote the YDA pathway. This study revealed that the mapkkk3 mapkkk5 double mutant showed reduced phosphorylation of MPK3/MPK6 upon treatment with conserved microbial molecules called PAMPs, suggesting that MAPKKK3/MAPKKK5 function upstream of MKK4/MKK5 to form a cascade in plant immunity. The YDA cascade shares the same MAPKK and MAPK as the MKK4/MKK5-MPK3/MPK6 cascade in plant immunity. Interestingly, loss of YDA or blocking BR signaling leads to increased elicitor-induced activation of MPK3/MPK6. On the other hand, development defects caused by silencing of YDA are suppressed in the mapkkk3 mapkkk5 double mutant. These data suggest that there are antagonistic interactions between the two MAP kinase cascades in stomatal development and plant immunity. Disruption of the MEKK1-MKK1/MKK2-MPK4 cascade leads to immune receptor SUMM2-mediated immune responses. MEKK2, a close paralog of MEKK1, was previously shown to act as a positive regulator of SUMM2-mediated immunity. I discovered that this occurred due to the inhibition of the activity of MPK4 by MEKK2 as MEKK2 is able to inhibit the phosphorylation of MPK4 by MKK2 in vitro. Interestingly, activation of SUMM2-mediated defense responses in the mekk1, mkk1 mkk2 and mpk4 mutant plants leads to increased MEKK2 transcript levels, which contributes to positive feedback regulation of SUMM2-mediated immunity. MEKK2 arose from a recent duplication event resulting in the tandem gene repeat consisting of MEKK1, MEKK2 and MEKK3. My data suggest that MEKK2 underwent dramatic functional divergence from other MAPKKKs to gain the function as a negative regulator of MAP kinases, which is an interesting evolutionary event conferring novel biochemical mechanisms of the paralog. Taken together, the studies in my Ph.D. thesis provide new insights into the regulation of the two main MAP kinase cascades activated during plant immunity.Science, Faculty ofBotany, Department ofGraduat

Association of crumbs homolog-2 with mTORC1 in developing podocyte.

The evidence that gene mutations in the polarity determinant Crumbs homologs-2 (CRB2) cause congenital nephrotic syndrome suggests the functional importance of this gene product in podocyte development. Because another isoform, CRB3, was reported to repress the mechanistic/mammalian target of the rapamycin complex 1 (mTORC1) pathway, we examined the role of CRB2 function in developing podocytes in relation to mTORC1. In HEK-293 and MDCK cells constitutively expressing CRB2, we found that the protein localized to the apicolateral side of the cell plasma membrane and that this plasma membrane assembly required N-glycosylation. Confocal microscopy of the neonate mouse kidney revealed that both the tyrosine-phosphorylated form and non-phosphorylated form of CRB2 commence at the S-shaped body stage at the apicolateral side of podocyte precursor cells and move to foot processes in a capillary tuft pattern. The pattern of phosphorylated mTOR in developing podocytes was similar to that of CRB2 tyrosine phosphorylation. Additionally, the lack of a tyrosine phosphorylation site on CRB2 led to the reduced sensitivity of mTORC1 activation in response to energy starvation. CRB2 may play an important role in the mechanistic pathway of developing podocytes through tyrosine phosphorylation by associating with mTORC1 activation