Sinorhizobium meliloti -induced chitinase gene expression in Medicago truncatula ecotype R108-1: a comparison between symbiosis-specific classV and defence-related classIV chitinases

The Medicago truncatula (Gaertn.) ecotypes JemalongA17 and R108-1 differ in Sinorhizobium meliloti-induced chitinase gene expression. The pathogen-inducible classIV chitinase gene, Mtchit4, was strongly induced during nodule formation of the ecotype JemalongA17 with the S. meliloti wild-type strain 1021. In the ecotype R108-1, the S. meliloti wild types Sm1021 and Sm41 did not induce Mtchit4 expression. On the other hand, expression of the putative classV chitinase gene, Mtchit5, was found in roots of M. truncatula cv. R108-1 nodulated with either of the rhizobial strains. Mtchit5 expression was specific for interactions with rhizobia. It was not induced in response to fungal pathogen attack, and not induced in roots colonized with arbuscular mycorrhizal (AM) fungi. Elevated Mtchit5 gene expression was first detectable in roots forming nodule primordia. In contrast to Mtchit4, expression of Mtchit5 was stimulated by purified Nod factors. Conversely, Mtchit4 expression was strongly elevated in nodules formed with the K-antigen-deficient mutant PP699. Expression levels of Mtchit5 were similarly increased in nodules formed with PP699 and its parental wild-type strain Sm41. Phylogenetic analysis of the deduced amino acid sequences of Mtchit5 (calculated molecular weight = 41,810Da, isoelectric point pH7.7) and Mtchit4 (calculated molecular weight 30,527Da, isoelectric point pH4.9) revealed that the putative Mtchit5 chitinase forms a separate clade within classV chitinases of plants, whereas the Mtchit4 chitinase clusters with pathogen-induced classIV chitinases from other plants. These findings demonstrate that: (i) Rhizobium-induced chitinase gene expression in M. truncatula occurs in a plant ecotype-specific manner, (ii) Mtchit5 is a putative chitinase gene that is specifically induced by rhizobia, and (iii) rhizobia-specific and defence-related chitinase genes are differentially influenced by rhizobial Nod factors and Kantigen

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

Studies of pathogenesis-related proteins in the strawberry plant: partial purification of a chitinase-containing protein complex and analysis of an osmotin-like protein gene

Plant chitinases and osmotin-like proteins (OLPs) are both pathogenesis-related (PR) proteins, which are implicated in plant responses to pathogen attacks and environmental stresses. In this dissertation, a chitinase-containing protein complex was purified to near homogeneity from strawberry leaf extracts. This protein complex contained at least five different chitinase molecules as revealed by activity gel assays. A previous study showed that winter rye leaves contain seven protein complexes, which consist of various combinations of a chitinase, two glucanase-like proteins (GLPs) and a thaumatin-like protein (TLP). Western blot analysis of the strawberry chitinase complex, however, did not detect the presence of any GLP or TLP in the complex. The second part of this dissertation research dealt with studies of strawberry OLP genes. A genomic clone containing an OLP gene, designated FaOLP2, was isolated and completely sequenced. FaOLP2 contains no intron, and has a potential to encode a precursor protein of 229 amino acid residues with a 27-amino acid signal peptide at the N-terminus. Southern blot analysis showed that FaOLP2 represents a small multi-gene family. The expression of FaOLP2 in different strawberry organs was analyzed using real-time PCR. The result showed that FaOLP2 expressed at different levels in leaves, crowns, roots, green fruits and ripe red fruits. Furthermore, the expression of FaOLP2 under different abiotic stresses was analyzed at different time points. All of the three tested abiotic stimuli, abscisic acid, salicylic acid and mechanical wounding, triggered significant induction of FaOLP2 within 2-6 h post-treatment. Comparing the three stimuli, FaOLP2 was more prominently induced by salicylic acid than by abscisic acid or mechanical wounding. The positive responses of FaOLP2 to these stress factors suggested that FaOLP2 may be involved in the protection of strawberry against pathogen attacks and against osmotic-related stresses. In addition to FaOLP2, the expression of a previously cloned OLP gene (FaOLP1) upon fungal infection was examined at different time points post-infection. Each of the two tested fungal species, Colletotrichum fragariae and Colletotrichum acutatum, triggered a substantial induction of FaOLP1 at 24-48 h post-inoculation, indicating that FaOLP1 could be involved in strawberry defense against fungal infection

Transformation of tobacco with a lupin chitinase gene under control of a stress inducible promoter

Chitinases are a diverse family of proteins occurring in plants. Their function varies considerably, with certain chitinases having been associated with development. The majority however, are pathogenesis related (PR) proteins that have been shown to play a role during plant pathogen interactions. This has lead to many investigations on the use of chitinases in providing transgenic disease resistance. These studies are usually done using a constitutive expression system. This however stands in contrast with the natural defense system were PR gene expression is usually only upregulated when the plant is exposed to abiotic and/or biotic stress factors. The constitutive expression is therefore not ideal as it increases ‘cost’ penalties due to the energy being spent expressing the gene. In this study however, an inducible expression system was applied using a stress inducible promoter AtGSTF6 derived from Arabidopsis thaliana, to drive Lupinus albus IF3 chitinase expression when the plants are under pathogen attack. The construct AtGSTF6-IF3 was inserted into the binary vector pCAMBIA 2300 and transformed into Nicotiana Tabacum cv JR6 by Agrobacterium-mediated transformation. To demonstrate the functionality of such a construct, an expression study was done on transgenic N. Tabacum to determine transcription and in vitro chitinase enzyme activity. The data revealed that IF3 chitinase gene transcription from lupin plants was achieved in N. Tabacum. Nine of the twelve lines that tested positive for chitinase gene transcription after hydrogen peroxide treatment, showed increased chitinase activity. With the success of showing increased chitinase activity, these lines were subjected to a detached leaf assay with Rhizoctonia solani AG2, which causes leaf target spot disease. The assay showed that six of the nine lines identified as having increased chitinase activity showed reductions in lesion areas. More specifically, three of the four lines showing more than a five-fold increase in chitinase activity compared to the untransformed N. Tabacum, showed significant lesion reduction. The AtGSTF6-IF3 construct can therefore be recommended to increase disease resistance in N. Tabacum towards Rhizoctonia solani AG2 after showing both expression and increased disease resistance in certain transgenic lines. CopyrightDissertation (MSc)--University of Pretoria, 2010.Plant Scienceunrestricte