| 研究概要 |
My JSPS project "Transcriptional dissection of the parasitic plant Striga asiatica in rice" aims to identify genes critical for Striga virulence by using transcriptional and genetic resources. In order to find these genes, we proposed a two-step strategy : In the first stage we would generate and analyse RNA sequencing (RNA Seq) data of in vitro grown S. asiatica treated with the haustorium inducing factor DMBQ. In the second stage we would perform RNA Seq on laser-microdissected Striga infection sites. We refrained from the initial idea to use DMBQ, because recent unpublished results by colleagues indicate significant morphological differences between DMBQ induced haustoria and host-induced haustoria in Phtheirospermum japonicum. P. japonicum is a related facultative hemi-parasite and used as a model species to study Striga-host interactions. We therefore decided to isolate Striga RNA directly from infected rice plants. This also allowed us to compare S. asiatica transcript data with
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already available transcript data for S. hermonthica and P. japonicum (Yoshida et al., in prep., Ishida et al., in prep.). In order to get statistical relevant data, we prepared three libraries of three independent infections, together with one library of above grown Striga tissue. We used the later one, together with two previously generated libraries from in vitro grown root and leaf tissue as control conditions for "non-pathogenic" gene expression. Principle component analysis revealed that all three biological replicates of Striga 7 dpi samples group closely together, and were distinct from non-pathogenic tissue. Secreted Striga proteins are potentially in direct contact with host cells, and therefore promising virulence factors. The current version of the S. asiatica genome encodes for 36,319 proteins, of which about 800 are predicted to be secreted, contain an N-terminal signalpeptide and have no transmembrane domain or GPI anchor. Roughly one half (398) of genes coding for these putative secreted proteins are at least 2-fold higher expressed in pathogenic Striga tissue compared to non-pathogenic Striga tissue. 77 of these genes are shared in all infection stages and both species and include one family of genes coding for Plant invertase/ Pectin-methyl-esterase Inhibitor (PMEI) domain containing proteins. Homologs of this family were found in S. asiatica, S. hermonthica and P. japonicum, but not in non-parasitic plans. All family members are highly up-regulated at late stages of infection (7 dpi) in all three species. Secretion of one family member was validated by translational RFP fusions, which showed apoplastic localization, when transiently expressed in N. benthamiana leaves. All S. asiatica members of this family were cloned into binary vectors downstream of the 35S promotor with or without C-terminal RFP tag. Arabidopsis thaliana plants were dipped in Agrobacteria containing those vectors to generate stable transgenic plants. These plants will be used in future to study the function of these proteins. Further experiments will include P. japonicum homologs, because transient transformation protocols for P. japonicum exist. At the moment, I am also adapting protocols for laser-microdessection (LMD) used on P. japonicum to S. asiatica. Sample preparation has improved significantly and first samples for RNA isolation were obtained. 隠す
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