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Mi-1.2, an R gene for aphid resistance in tomato, has direct negative effects on a zoophytophagous biocontrol agent, Orius insidiosus.

Pallipparambil GR, Sayler RJ, Shapiro JP, Thomas JM, Kring TJ, Goggin FL - J. Exp. Bot. (2014)

Bottom Line: Molecular gut content analysis confirmed that adults and immatures of O. insidiosus feed on both resistant (Mi-1.2+) and susceptible (Mi-1.2-) genotypes, and bioassays suggest that resistance does not affect oviposition rates, plant sampling, or prey acceptance by O. insidiosus adults.These results demonstrate a direct negative impact of R-gene-mediated host plant resistance on a non-target beneficial species, and reveal that Mi-mediated resistance can impact organisms that do not feed on phloem sap.These results suggest that the mode of action and potential ecological impacts of Mi-mediated resistance are broader than previously assumed.

View Article: PubMed Central - PubMed

Affiliation: 319 Agricultural Building, Department of Entomology, University of Arkansas, Fayetteville, AR 72701, USA.

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Related in: MedlinePlus

Localization of Mi-1.2 transcripts in tomato tissues. (A) RT-PCR was used to assess expression of Mi-1.2 in epidermis (E), mesophyll (M), and phloem (P) samples harvested by LCM from the petioles of 143-25 (Mi-1.2+) and the susceptible cultivar Moneymaker (Mi-1.2–). Primers to the ubiquitously-expressed housekeeping gene Ribosomal protein L2 (Rpl2) were used to confirm good cDNA synthesis, and primers that amplify phloem-specific transcripts (Prosystemin and G2-like transcription factor) were used to confirm that the microdissection procedure adequately separated vascular and non-vascular tissues. (B) The G2-like transcription factor (G2-LTF) primers were also used to confirm the absence of genomic DNA in the samples, because the primers flank an intron and generate different amplicon sizes from genomic DNA (G) versus our cDNA templates. The gel lanes in this figure were spliced and rearranged to conveniently compare gene expression among the targeted tissues.
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Figure 5: Localization of Mi-1.2 transcripts in tomato tissues. (A) RT-PCR was used to assess expression of Mi-1.2 in epidermis (E), mesophyll (M), and phloem (P) samples harvested by LCM from the petioles of 143-25 (Mi-1.2+) and the susceptible cultivar Moneymaker (Mi-1.2–). Primers to the ubiquitously-expressed housekeeping gene Ribosomal protein L2 (Rpl2) were used to confirm good cDNA synthesis, and primers that amplify phloem-specific transcripts (Prosystemin and G2-like transcription factor) were used to confirm that the microdissection procedure adequately separated vascular and non-vascular tissues. (B) The G2-like transcription factor (G2-LTF) primers were also used to confirm the absence of genomic DNA in the samples, because the primers flank an intron and generate different amplicon sizes from genomic DNA (G) versus our cDNA templates. The gel lanes in this figure were spliced and rearranged to conveniently compare gene expression among the targeted tissues.

Mentions: Orius insidiosus does not feed from the phloem like other pests that are impacted by Mi-1.2. To detect low levels of Mi-1.2 expression and to discriminate this gene from its many close homologues in tomato, a combination of laser capture microdissection (LCM) and RT-PCR were used to determine the localization of Mi-1.2 transcripts in resistant plant tissues. Cryosectioning and LCM were used to dissect samples of epidermal, mesophyll, and phloem tissues from tomato plants that lack Mi-1.2 (cv. Moneymaker), and isogenic transgenic plants (143-25) that express Mi-1.2 driven by its native promoter in the same genetic background (cv. Moneymaker). RT-PCR performed on RNA from the harvested tissue provided evidence that the Mi-1.2 gene is transcribed in all three tissue types (Fig. 5A). The experiment was repeated three times using separate plants. In 143-25, Mi-1.2 transcripts were detected in all three samples of epidermis tissue, two out of three samples of mesophyll tissue, and one out of three samples of phloem tissue. Primers to detect Mi-1.2 expression did not generate PCR products from any of the tissue samples from susceptible plants, indicating that our primers did not amplify any of the Mi-1 homologues present in this genetic background. The presence of cDNA in all samples was confirmed by amplifying transcripts encoding the ribosomal protein RPL2 (Fig. 5A). Primers for prosystemin and a G2-like transcription factor (G2-LTF), which are expressed specifically in the phloem, detected transcripts in the phloem samples but not in any of the epidermis or mesophyll samples, providing evidence that the LCM sectioning adequately isolated the tissue types (Fig. 5A). The prosystemin primers generated PCR products in two out of the three phloem samples, while G2-LTF expression was detected in all three phloem samples. Results for the G2-LTF primers also indicated that our samples were free of contamination from genomic DNA. This primer set flanks an intron and generates a PCR product of ~500bp from genomic DNA samples, whereas it only yields a single amplicon of 186bp from our cDNA samples (Fig. 5B).


Mi-1.2, an R gene for aphid resistance in tomato, has direct negative effects on a zoophytophagous biocontrol agent, Orius insidiosus.

Pallipparambil GR, Sayler RJ, Shapiro JP, Thomas JM, Kring TJ, Goggin FL - J. Exp. Bot. (2014)

Localization of Mi-1.2 transcripts in tomato tissues. (A) RT-PCR was used to assess expression of Mi-1.2 in epidermis (E), mesophyll (M), and phloem (P) samples harvested by LCM from the petioles of 143-25 (Mi-1.2+) and the susceptible cultivar Moneymaker (Mi-1.2–). Primers to the ubiquitously-expressed housekeeping gene Ribosomal protein L2 (Rpl2) were used to confirm good cDNA synthesis, and primers that amplify phloem-specific transcripts (Prosystemin and G2-like transcription factor) were used to confirm that the microdissection procedure adequately separated vascular and non-vascular tissues. (B) The G2-like transcription factor (G2-LTF) primers were also used to confirm the absence of genomic DNA in the samples, because the primers flank an intron and generate different amplicon sizes from genomic DNA (G) versus our cDNA templates. The gel lanes in this figure were spliced and rearranged to conveniently compare gene expression among the targeted tissues.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4286404&req=5

Figure 5: Localization of Mi-1.2 transcripts in tomato tissues. (A) RT-PCR was used to assess expression of Mi-1.2 in epidermis (E), mesophyll (M), and phloem (P) samples harvested by LCM from the petioles of 143-25 (Mi-1.2+) and the susceptible cultivar Moneymaker (Mi-1.2–). Primers to the ubiquitously-expressed housekeeping gene Ribosomal protein L2 (Rpl2) were used to confirm good cDNA synthesis, and primers that amplify phloem-specific transcripts (Prosystemin and G2-like transcription factor) were used to confirm that the microdissection procedure adequately separated vascular and non-vascular tissues. (B) The G2-like transcription factor (G2-LTF) primers were also used to confirm the absence of genomic DNA in the samples, because the primers flank an intron and generate different amplicon sizes from genomic DNA (G) versus our cDNA templates. The gel lanes in this figure were spliced and rearranged to conveniently compare gene expression among the targeted tissues.
Mentions: Orius insidiosus does not feed from the phloem like other pests that are impacted by Mi-1.2. To detect low levels of Mi-1.2 expression and to discriminate this gene from its many close homologues in tomato, a combination of laser capture microdissection (LCM) and RT-PCR were used to determine the localization of Mi-1.2 transcripts in resistant plant tissues. Cryosectioning and LCM were used to dissect samples of epidermal, mesophyll, and phloem tissues from tomato plants that lack Mi-1.2 (cv. Moneymaker), and isogenic transgenic plants (143-25) that express Mi-1.2 driven by its native promoter in the same genetic background (cv. Moneymaker). RT-PCR performed on RNA from the harvested tissue provided evidence that the Mi-1.2 gene is transcribed in all three tissue types (Fig. 5A). The experiment was repeated three times using separate plants. In 143-25, Mi-1.2 transcripts were detected in all three samples of epidermis tissue, two out of three samples of mesophyll tissue, and one out of three samples of phloem tissue. Primers to detect Mi-1.2 expression did not generate PCR products from any of the tissue samples from susceptible plants, indicating that our primers did not amplify any of the Mi-1 homologues present in this genetic background. The presence of cDNA in all samples was confirmed by amplifying transcripts encoding the ribosomal protein RPL2 (Fig. 5A). Primers for prosystemin and a G2-like transcription factor (G2-LTF), which are expressed specifically in the phloem, detected transcripts in the phloem samples but not in any of the epidermis or mesophyll samples, providing evidence that the LCM sectioning adequately isolated the tissue types (Fig. 5A). The prosystemin primers generated PCR products in two out of the three phloem samples, while G2-LTF expression was detected in all three phloem samples. Results for the G2-LTF primers also indicated that our samples were free of contamination from genomic DNA. This primer set flanks an intron and generates a PCR product of ~500bp from genomic DNA samples, whereas it only yields a single amplicon of 186bp from our cDNA samples (Fig. 5B).

Bottom Line: Molecular gut content analysis confirmed that adults and immatures of O. insidiosus feed on both resistant (Mi-1.2+) and susceptible (Mi-1.2-) genotypes, and bioassays suggest that resistance does not affect oviposition rates, plant sampling, or prey acceptance by O. insidiosus adults.These results demonstrate a direct negative impact of R-gene-mediated host plant resistance on a non-target beneficial species, and reveal that Mi-mediated resistance can impact organisms that do not feed on phloem sap.These results suggest that the mode of action and potential ecological impacts of Mi-mediated resistance are broader than previously assumed.

View Article: PubMed Central - PubMed

Affiliation: 319 Agricultural Building, Department of Entomology, University of Arkansas, Fayetteville, AR 72701, USA.

Show MeSH
Related in: MedlinePlus