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" (2007)

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