Studies in flowering plants have provided a wealth of information on pathogen recognition, signal transduction and the activation of defense responses. However, very little is known about the immune system of the phylogenetically ancient moss Physcomitrella patens. Mosses represent some of the earliest land plants and are thus in an ideal evolutionary position to provide information on the evolution of plant innate immune systems. Furthermore, Physcomitrella has the unique ability to be genetically manipulated using targeted gene replacements through homologous recombination.
Using this emerging model system, we identify and create targeted knock out of nine Physcomitrella homologs of defense related Arabidopsis genes. The knock-out lines are assessed for altered immune responses to a range of different pathogens.
We find that at least one Physcomitrella mitogen activated protein kinase (MPK), PpMPK4A is required for proper innate immune responses. This is a primary example of a single, non-redundant plant MPK essential for immunity without any other apparent phenotypes associated with the corresponding null-mutant. We show that PpMPK4A is phosphorylated in response to microbe associated molecular patterns (MAMPs) including fungal chitin and bacterial MAMPs. The knock out of PpMPK4A renders the moss more susceptible to the pathogenic fungi Botrytis cinerea and Alternaria brassicicola and fails to accumulate several defense related transcripts and ROS production upon treatment with fungal chitosan. While related MPKs in the higher plant model Arabidopsis thaliana are activated both by pathogen inoculation and by abiotic stress, we did not detect activation of PpMPK4A or any other Physcomitrella MPK by several abiotic stresses. Signal transduction via PpMPK4A may therefore be specific to MAMP-triggered immunity, and the moss may use other signaling components to respond to abiotic stresses.
In addition, a Physcomitrella knock-out of a homolog of the autophagy related gene ATG5 provides the first analysis of autophagy in non-vascular plants. PpATG5 knock-out mutants show clear signs of autophagy deficiency, such as early senescence and sensitivity to nutrient deprivation. We also show that PpATG5 is required for the defense against a Sordariomycetes fungus.