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Co-immunoprecipitation (Co-IP) in Nicotiana benthamiana followed by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS)

ABSTRACT: Ralstonia solanacearum species complex causes bacterial wilt in variety crops. Tomato cultivar Hawaii 7996 is a widely used resistance resource, however, the resistance is evaded by virulent strains with underlying mechanisms still unknown. Here, we reported that phylotype II strain ES5-1 can overcome Hawaii 7996 resistance. RipV2, a type Ⅲ effector specifically to phylotype Ⅱ strains, is vital in overcoming tomato resistance. RipV2, which encodes a novel E3 ubiquitin ligase, suppresses immune responses and TNL-mediated cell death. Tomato helper NLR N requirement gene 1 (NRG1), enhanced disease susceptibility 1 (EDS1), and senescence-associated gene 101b (SAG101b) were identified as RipV2 target proteins. RipV2 is essential for ES5-1 virulence in Hawaii 7996 but not in SlNRG1-silenced tomato, demonstrating SlNRG1 to be the bona fide RipV2 virulence target. Our results dissected the mechanisms of RipV2 in disrupting immunity and highlighted the importance of converged immune components in conferring bacterial wiltresistance.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Nicotiana Benthamiana

TISSUE(S): Plant Cell, Leaf

DISEASE(S): Disease Free

SUBMITTER: Qi peipei

LAB HEAD: Li Bo

PROVIDER: PXD054061 | Pride | 2024-07-20

REPOSITORIES: Pride

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Project description:Metastatic disease remains one of the most urgent clinical challenges accounting for over 90% of cancer-related deaths. Yet, the identification of novel therapeutic targets to fight or prevent metastatic disease has been hampered by the limited availability of clinically relevant mouse models of metastasis formation. To address this caveat, we developed a novel preclinical mouse model of spontaneous metastatic breast cancer that recapitulates the key biological events of the metastatic cascade and mimics the clinical course of metastatic disease in humans. Exploiting the conditional K14cre;CdhF/F;Trp53F/F mouse model of de novo mammary tumor formation, we orthotopically transplanted K14cre;CdhF/F;Trp53F/F derived mouse invasive lobular carcinoma (mILC) fragments into mammary glands of wild-type syngeneic hosts. Once recipient mammary tumors were established, we mimicked the clinical setting and performed a mastectomy. Following surgery, recipient mice eventually succumbed to wide-spread clinically overt metastatic disease in lymph nodes, lungs and gastrointestinal tract. Using aCGH analyses, we explored the relationship between the genomic profiles of mammary donor tumors and paired recipient outgrowths and observed a strong correlation, indicating that the genomic profile of the parental K14cre;CdhF/F;Trp53F/F mILC is highly conserved in recipient mammary tumors. To investigate the genomic relationship between recipient mammary tumors and their metastases, we examined the correlation structure of genomic profiles derived from paired sets of primary tumors and metastases. Genomic profiles of clonally-related recipient mammary tumors were highly conserved in local and distant metastases, indicating that few genomic alterations occur during transition from a primary tumor to a distant site. To more thoroughly examine potential site-specific genomic alterations, we constructed so-called ‘delta-profiles’ by calculating the difference between the genomic profile of a recipient mammary tumor and its paired lymph node- and lung metastasis. Site-specific recurrent alterations were not observed in lymph node nor lung metastases. Taken together, these data show that genomic profiles of metastases are highly similar to those of parental recipient tumors and that, if changes occurred, they did not recur in different independent samples. We performed aCGH analyses on DNA isolated from K14cre;Cdh-/-;Trp53-/- derived donor mILCs (n=3) and their recipient mammary tumor outgrowths (n=10). Furthermore, we also analyzed genomic profiles derived from lung (n=10), tumor-draining (n=7) and distant lymph node metastases (n=5) isolated from the same recipient mice. DNA from each of these samples was hybridized against related donor splenic DNA.

Project description:A comparative study ware made to know the Early Blight resistance mechanism between resistant and susceptible plants at vegetative stage. Transcriptional changes during compatible and resistant reaction between A. solani and tomato genotypes have been identified using Affymetrix tomato gene chips. Results were confirmed by reverse transcription-polymerase chain reaction with the samples collected after 24hrs of inoculation from inoculated and control susceptible and resistant genotypes. The number of up and down regulated genes in three different combinations of treatments i.e. Early Blight Resistant Treated and Control (EBTT and EBTC) and Early Blight Suceptible Treated and Control (EBST and EBSC) has observed and compared. In EBST vs. EBSC condition, 3478 genes are up regulated and 2663 are down regulated. Similarly when analysis is done between EBST and EBRT, 2475 genes are up regulated and 3662 are down regulated. Total 3442 genes are up regulated and 2695 genes are found to be down regulated in resistant line of tomato for early blight when the analysis is done for EBRT Vs. EBRC. These genes, that are induced in the resistant genotype EC-520061(Solanum habrochaites , wild) of tomato, after Alternaria solani infection, have been classified in to following categories, response to stress, cellular component, Metabolic activity, regulation of transcription, regulation of translation, and few were unknown. Data from this study are a resource for understanding the mechanism, which works for resistance against pathogen of early blight in tomato, which is critical and will be useful in improving this trait through transgenic approach or gene silencing. Tomato plants were exposed to A. solani spores and after 24 hrs (at the stage of penetration of tissues with fungal hyphae) samples were collected from both susceptible and resistant genotypes for RNA extraction and hybridization on Affymetrix chips. To study the molecular mechanism of biotic stress resistance, transcripts of both the genotypes were compared in response to A. solani.

			Action	DRS
	C403A-FLAG.pep.xml	Pepxml
	C403A-FLAG.pro.xml	Xml
	C403A-FLAG.raw	Raw
	CK.pep.xml	Pepxml
	CK.pro.xml	Xml

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Co-immunoprecipitation (Co-IP) in Nicotiana benthamiana followed by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS)

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