Gene induction during plant-microbe interactions : the role of chitinases during fungal infection and the investigation of mycorrhiza-induced genes in the model plant "M. truncatula"

In this thesis, the model legume Medicago truncatula was used for research on plant-microbe
interactions. Unlike most other plants, legumes are able to form two distinct root symbioses.
Together with soil-borne fungi of the Glomeromycota, they form the arbuscular mycorrhiza
and with rhizobial bacteria, they form nitrogen fixing root nodules. Here, plant responses to
these symbiotic microbes were investigated, and compared to the plant's defence response
against antagonistic microbes.
Plant chitinases have an important role in the interplay between plants and microbes; they
have been shown to act as defence-related antifungal enzymes, but they seem to be
involved in symbiotic processes as well. In previous work, genes encoding eight different
chitinases were identified in M. truncatula. The main part of this thesis is dedicated to an indepth
study of these genes with regard to their regulation and function. Additionally, the
expression patterns of genes that are related to the arbuscular mycorrhizal interaction of M.
truncatula were analysed in order to further explore this important symbiosis.
In roots of the M. truncatula ecotype R108-1, expression of the gene Mtchit5, encoding a
class V chitinase, was induced during nodule formation after infection with wild type rhizobia,
but not in response to pathogenic fungi or arbuscular mycorrhizal fungi. Mtchit5 transcripts
were first detectable in roots forming nodule primordial and accumulated during nodule
ripening. The gene was induced in response to purified Nod factors and also in ineffective
white nodules formed by a mutant rhizobial strain. Phylogenetic analysis of the deduced
amino acid sequence revealed that the putative Mtchit5 chitinase forms a separate clade
within class V chitinases of plants. These results, together with the additional finding that
Mtchit5 expression is high in flowers, indicate that Mtchit5 is a putative early nodulin that is
specifically induced by rhizobia in roots and may have a function in plant developmental
processes.
The gene Mtchit4, encoding a class IV chitinase, is induced during infection by pathogenic
fungi in roots of M. truncatula but not during mycorrhiza formation. During nodule formation,
Mtchit4 was strongly induced only in the M. truncatula ecotype Jemalong A17 after infection
with wild-type rhizobium strains. Its expression was elevated in nodules formed with a Kantigen
deficient rhizobium mutant, but not in response to purified Nod factors. The putative
Mtchit4 chitinase is closely related to pathogenesis-related class IV chitinases from other
plants, and it is assumed that Mtchit4 is a pathogenesis related protein. This is supported by
an additional study that revealed a low overall expression of Mtchit4 throughout the plant,
independent of the plant’s symbiotic status, and an in silico analysis of the Mtchit4 promoter
sequence, which contains a variety of putative cis-elements related to plant defence.
The expression of two genes encoding class I chitinases, Mtchit1a and Mtchit1c, were
compared to the expression of Mtchit4 in leaves, roots and flowers and after infection with a
mycorrhizal fungus. In contrast to the constitutively expressed Mtchit1c, the expression of
Mtchit1a was similar in leaves or roots but low in flowers. Both chitinase genes were not
affected by mycorrhizal infection. The differential expression patterns, together with
sequence data and in silico promoter analyses, suggest that these genes encode
pathogenesis related chitinases, that are specifically regulated in response to infection by
pathogenic fungi.
Mtchit3-3 is a class III chitinase gene that was specifically induced in mycorrhizal roots. The
Mtchit3-3 promoter directs reporter gene expression to arbuscule containing cells, which is
consistent with mycorrhiza-related elements found in the promoter sequence. Disruption of
the Mtchit3-3 gene expression in root organ cultures stimulated spore germination of
mycorrhizal fungi and in one fungal strain resulted in a higher probability of root colonization
and spore formation. No effect on the abundance of arbuscules within colonized roots
became apparent. Mtchit3-3-GFP fusion constructs revealed that the putative signal peptide
could direct the Mtchit3-3 protein to the apoplast. It is suggested that the chitinase Mtchit3-3
is enzymatically active and might act on chitin in the fungal cell wall or fungal chitin-related
signals during the symbiosis and it may be involved in communication processes between
plant and AM.
The class III chitinase genes Mtchit3-1 and Mtchit3-4 are induced in response to infection by
pathogenic fungi in roots of M. truncatula. Mtchit3-4, but not Mtchit3-1, was also slightly
induced during mycorrhiza formation. According to their gene and protein structures in
comparison to chitinases in other plant species and additional in silico promoter analyses, it
is proposed that Mtchit3-1 is a pathogenesis related chitinase while Mtchit3-4 may be related
in a general way to fungal infections.
The functionality of the arbuscular mycorrhizal symbiosis was measured by comparing the
plant’s nutritional status and growth response of three mycorrhizal fungi from two different
phylogenetic taxa, namely Glomus intraradices, Glomus mosseae and Scutellospora
castanea. Mycorrhiza formation enhanced biomass accumulation and nutritional status of the
plants in each case, although the response was not related to the colonization degree.
To supplement the expression data of chitinase genes in relation to different fungal
infections, the expression was measured in the roots of M. truncatula plants colonized with
the three AMF. In addition, a selected set of other symbiosis related genes were tested that
responded differently to the AMF colonization. It can be concluded that a subset of the genes
that respond to colonization by the two Glomus species also responded to at least one
fungus from the Gigasporaceae. These data indicate that different genes showing arbusculespecific
gene expression in colonized roots are regulated by different mechanisms,
depending on the fungal partner.