Plant roots are a wonder. Whilst roots are taking up water and nutrients from the soil, they are exposed to all sorts of microbes in the soil. Some microbes are good and promote plant growth, whilst others attack the roots and cause diseases. In defense of a plant pathogen attack, plants can have multiple defensive mechanisms. One of them is changing the root cell walls to create a “barricade” to prevent the pathogen from entering.
Dr Romain Castilleux from the Swedish University of Agricultural Sciences and colleagues from four French institutes reported in 2019 that extensins, a group of cell wall proteins, are important for providing the model plant, Arabidopsis thaliana against the plant pathogen, Phytophthora parasitica. In the following year, Drs Li Tan and Andrew Mort commented on the research methods used for measuring extensins levels in the plant roots by Castilleux and colleagues.In response, Castilleux and colleagues adjusted their approach and now report new results that continue to support their research findings about extensins. This story of investigating the unknown mechanisms behind cell wall assembly and scientists debating research methods demonstrates how research works.
Whilst almost everyone can remember the plant cell illustration in biology class, plant cell walls were usually just one line, surrounding the “exciting” cytoplasm, mitochondrion, ribosome and such. However, plant cell walls represent one of the most complex structural networks in nature. The nanoscale network primarily consists of polysaccharide polymers such as cellulose, hemicellulose, and pectin, but often includes glycoproteins and lignin. Extensins are cell wall glycoproteins that are involved in embryo development, root hair growth, and cell wall assembly and structure.
In 2018, Castilleux and colleagues reviewed the role of cell wall extensins in plant-microbe interactions and hypothesised that extensins would cross-link in response to the pathogenic infection in order to limit disease entry. The researchers highlighted the potential of using antibodies to detect extensin epitopes (specific pieces of the antigen to which an antibody binds to). Then, the accumulation of extensins can be visualised using confocal laser microscopy on cut root sections. The production of extensins was suggested to rely on arabinosylation (addition of arabinose, a monosaccharide containing five carbon atoms and an aldehyde functional group) allowing the formation of cross-links essential for cell wall assembly and organisation. At the end of the review, Castilleux and colleagues suggested that arabinosylation of extensins is crucial in plant immune response against soilborne pathogens. This led to their study in 2019, testing this hypothesis.
Castilleux and colleagues infected Arabidopsis mutants that were altered to have a normal immune response or none, and have different levels of extensin glycosylation (polysaccharide attachment to extensin protein) with the soilborne pathogen, Phytophthora parasitica to test if extensins are important in the immune response. The researchers used immunofluorescence microscopy on 9-day-old A. thaliana roots using anti-extensin-specific antibodies.
“Our findings suggest that glycosylation of extensins, and more specifically their arabinosylation, is essential for cell wall remodelling during the immune response of A. thaliana root cells”, Castilleux and colleagues concluded their study in 2020.
In response to this study, Tan and Mort wrote a commentary, expressing some concerns about the methods and conclusion of the study, especially whether the used epitopes are truly specific for extensin arabinosides.
“Although we can be persuaded that extensins were highly expressed in root tips both before and after elicitation, the complicated antibody recognition patterns and pattern changes for each mutant after elicitation make it hard to explain the data, thus weakening the actual evidence”, Tan and Mort wrote.
They added that it would be very interesting to experimentally test if there are higher levels of extensin intermolecular cross-linking in response to a microbe attack.
In response, Castilleux and colleagues provided evidence of the cross-linking response by using new A. thaliana mutants (PEROXIDASES 33 and 34) that could catalyze the cross-linking of extensins. No extensins could be visualised on the mutant that had no immune response but high amounts of extensis could be seen in the cross-linking mutant.
“This suggests that the mutation of the genes encoding PEROXIDASES 33 and 34 impacts detection of the extensin LM1 epitope (and possibly the formation of extensin cross-links) in response to [immune response elicitation]”, Castilleux and colleagues wrote.
Whilst there are many technical challenges for uncovering what mechanism (arabinosylation, glycosylation, cross-linking) is crucial for extensin production/incorporation into the cell wall, Castilleux has shown the potential of using mutant plants and immunolabeling.
“In conclusion, extensins appear to play an important role in root defence, with arabinosylation essential for their correct function, probably through a controlled crosslinking catalysed by specific cell wall peroxidases.”