Download Deciphering the interplay between cell wall integrity, sensing and

Deciphering the interplay between cell wall integrity, sensing and
immunity
This summer I was very fortunate to have the opportunity to work in the group of Professor Cyril
Zipfel at The Sainsbury Laboratory, Norwich. Cyril’s lab primarily seeks to understand the
molecular basis of pattern-triggered immunity (PTI); an area of plant-microbe interactions I find
particularly exciting, especially given the recent successful transfer of pattern recognition
receptors between the monocot/eudicot clades. Whilst many of the elicitors studied by the lab
are derived from pathogens my project focus was attempting to understand how plant cells
perceive disruption of their cell wall.
Plant cells are surrounded by a heterogeneous extracellular matrix of polymers known as the cell
wall. The physical properties of the wall direct cell growth, as well protecting the cell against
pathogens and abiotic stresses. Perturbations in the integrity of the cell wall can prove fatal to
the cell and are indicative of developmental errors, abiotic stresses or pathogen mediated cell
wall degradation. Thus, the ability of plants to actively monitor and respond to cell wall status
increases their chance of survival. While the mechanisms by which this is achieved has been
described in yeast we are only beginning to elucidate these mechanisms in plants.
The Zipfel lab found that many of the responses to pharmacological inhibition of cellulose
biosynthesis are shared with PTI in Arabidopsis thaliana. Wild-type plants exhibit ectopic
lignification, growth inhibition, jasmonic acid accumulation and defence gene expression. Using
a reverse genetic approach the lab identified an A. thaliana receptor-like kinase (RLK) mutant
with an impaired response to isoxaben (ISX), a chemical widely used to disrupt cellulose
biosynthesis in a controlled manner. Mutants in this RLK, tentatively named LRI (Leucine-rich
repeat RLK Required for ISX response), also exhibit an additional growth phenotype as their roots
display a distinct skewing to the left.
During my studentship in the Zipfel lab, I attempted to identify additional components of the LRI
signalling pathway. Prior to my arrival, T-DNA insertional lines were ordered in candidate genes
from the Nottingham Arabidopsis Stock Centre. Candidates were selected based on coexpression with LRI or LRI dependency for ISX-induced transcript regulation. The selection was
enriched in genes encoding RLKs and peptides, which may potentially act as co-receptors or
ligands, respectively. I proceeded to design primers for these T-DNA lines allowing me to PCRgenotype the lines, facilitating the generation of homozygous insertional mutants. These mutants
could then be assayed for an abnormal response to ISX treatment, indicating they may function
in the same pathway as LRI. Fortunately, homozygous mutant lines for several of the candidates
were already available in the lab allowing me to progress with these experiments. I measured
ISX-induced expression of 3 reporter genes using RT-qPCR, and quantified the root skewing
phenotype by growing the seedlings on vertical plates and measuring root angle using the
software ImageJ. Some of the candidates appear to show interesting responses and merit further
examination.
My experience in the Zipfel lab was extremely valuable; it was my first experience in molecular
biology and I thoroughly enjoyed it. I learned a huge amount over the summer; not only in the
lab, but also through the many seminars, lab meetings and journal clubs I was able to attend. I
would like to thank all the Zipfel lab for making me feel so welcome, especially Dr Dieuwertje Van
der Does who mentored me throughout. I am extremely grateful to the BSPP for generously
providing the funding and membership; I hope the invaluable experience I gained over the
summer will serve me well as I progress with my studies and pursue a career in molecular plant
biology.
Jack Rhodes
University of Bath