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&#13;\ninteractions. Unlike most other plants, legumes are able to form two distinct root symbioses.&#13;\nTogether with soil-borne fungi of the Glomeromycota, they form the arbuscular mycorrhiza&#13;\nand with rhizobial bacteria, they form nitrogen fixing root nodules. Here, plant responses to&#13;\nthese symbiotic microbes were investigated, and compared to the plant's defence response&#13;\nagainst antagonistic microbes.&#13;\nPlant chitinases have an important role in the interplay between plants and microbes; they&#13;\nhave been shown to act as defence-related antifungal enzymes, but they seem to be&#13;\ninvolved in symbiotic processes as well. In previous work, genes encoding eight different&#13;\nchitinases were identified in M. truncatula. The main part of this thesis is dedicated to an indepth&#13;\nstudy of these genes with regard to their regulation and function. Additionally, the&#13;\nexpression patterns of genes that are related to the arbuscular mycorrhizal interaction of M.&#13;\ntruncatula were analysed in order to further explore this important symbiosis.&#13;\nIn roots of the M. truncatula ecotype R108-1, expression of the gene Mtchit5, encoding a&#13;\nclass V chitinase, was induced during nodule formation after infection with wild type rhizobia,&#13;\nbut not in response to pathogenic fungi or arbuscular mycorrhizal fungi. Mtchit5 transcripts&#13;\nwere first detectable in roots forming nodule primordial and accumulated during nodule&#13;\nripening. The gene was induced in response to purified Nod factors and also in ineffective&#13;\nwhite nodules formed by a mutant rhizobial strain. Phylogenetic analysis of the deduced&#13;\namino acid sequence revealed that the putative Mtchit5 chitinase forms a separate clade&#13;\nwithin class V chitinases of plants. These results, together with the additional finding that&#13;\nMtchit5 expression is high in flowers, indicate that Mtchit5 is a putative early nodulin that is&#13;\nspecifically induced by rhizobia in roots and may have a function in plant developmental&#13;\nprocesses.&#13;\nThe gene Mtchit4, encoding a class IV chitinase, is induced during infection by pathogenic&#13;\nfungi in roots of M. truncatula but not during mycorrhiza formation. During nodule formation,&#13;\nMtchit4 was strongly induced only in the M. truncatula ecotype Jemalong A17 after infection&#13;\nwith wild-type rhizobium strains. Its expression was elevated in nodules formed with a Kantigen&#13;\ndeficient rhizobium mutant, but not in response to purified Nod factors. The putative&#13;\nMtchit4 chitinase is closely related to pathogenesis-related class IV chitinases from other&#13;\nplants, and it is assumed that Mtchit4 is a pathogenesis related protein. This is supported by&#13;\nan additional study that revealed a low overall expression of Mtchit4 throughout the plant,&#13;\nindependent of the plant’s symbiotic status, and an in silico analysis of the Mtchit4 promoter&#13;\nsequence, which contains a variety of putative cis-elements related to plant defence.&#13;\nThe expression of two genes encoding class I chitinases, Mtchit1a and Mtchit1c, were&#13;\ncompared to the expression of Mtchit4 in leaves, roots and flowers and after infection with a&#13;\nmycorrhizal fungus. In contrast to the constitutively expressed Mtchit1c, the expression of&#13;\nMtchit1a was similar in leaves or roots but low in flowers. Both chitinase genes were not&#13;\naffected by mycorrhizal infection. The differential expression patterns, together with&#13;\nsequence data and in silico promoter analyses, suggest that these genes encode&#13;\npathogenesis related chitinases, that are specifically regulated in response to infection by&#13;\npathogenic fungi.&#13;\nMtchit3-3 is a class III chitinase gene that was specifically induced in mycorrhizal roots. The&#13;\nMtchit3-3 promoter directs reporter gene expression to arbuscule containing cells, which is&#13;\nconsistent with mycorrhiza-related elements found in the promoter sequence. Disruption of&#13;\nthe Mtchit3-3 gene expression in root organ cultures stimulated spore germination of&#13;\nmycorrhizal fungi and in one fungal strain resulted in a higher probability of root colonization&#13;\nand spore formation. No effect on the abundance of arbuscules within colonized roots&#13;\nbecame apparent. Mtchit3-3-GFP fusion constructs revealed that the putative signal peptide&#13;\ncould direct the Mtchit3-3 protein to the apoplast. It is suggested that the chitinase Mtchit3-3&#13;\n&#13;\nis enzymatically active and might act on chitin in the fungal cell wall or fungal chitin-related&#13;\nsignals during the symbiosis and it may be involved in communication processes between&#13;\nplant and AM.&#13;\nThe class III chitinase genes Mtchit3-1 and Mtchit3-4 are induced in response to infection by&#13;\npathogenic fungi in roots of M. truncatula. Mtchit3-4, but not Mtchit3-1, was also slightly&#13;\ninduced during mycorrhiza formation. According to their gene and protein structures in&#13;\ncomparison to chitinases in other plant species and additional in silico promoter analyses, it&#13;\nis proposed that Mtchit3-1 is a pathogenesis related chitinase while Mtchit3-4 may be related&#13;\nin a general way to fungal infections.&#13;\nThe functionality of the arbuscular mycorrhizal symbiosis was measured by comparing the&#13;\nplant’s nutritional status and growth response of three mycorrhizal fungi from two different&#13;\nphylogenetic taxa, namely Glomus intraradices, Glomus mosseae and Scutellospora&#13;\ncastanea. Mycorrhiza formation enhanced biomass accumulation and nutritional status of the&#13;\nplants in each case, although the response was not related to the colonization degree.&#13;\nTo supplement the expression data of chitinase genes in relation to different fungal&#13;\ninfections, the expression was measured in the roots of M. truncatula plants colonized with&#13;\nthe three AMF. In addition, a selected set of other symbiosis related genes were tested that&#13;\nresponded differently to the AMF colonization. It can be concluded that a subset of the genes&#13;\nthat respond to colonization by the two Glomus species also responded to at least one&#13;\nfungus from the Gigasporaceae. These data indicate that different genes showing arbusculespecific&#13;\ngene expression in colonized roots are regulated by different mechanisms,&#13;\ndepending on the fungal partner.

Topic: Legume Nitrogen Fixing Symbiosis
Subfield: Plant Science
Field: Agricultural and Biological Sciences
Domain: Life Sciences