Virus and fungal resistance: from laboratory to field

Virus and fungal resistance traits are important targets in the genetic engineering of agricultural and horticultural crops. We have engineered resistance against potato virus X in important commercial potato cultivars. Four years of field trials with resistant potatoes have demonstrated the commercial feasibility of improving potato cultivars by selectively adding new traits while preserving intrinsic properties. In our pursuit for a broad resistance against fungi we have focused on the exploitation of genes encoding antifungal proteins. We present results demonstrating the antifungal effect of some of these proteins in vitro , as well as the synergy between specific chitinases and β-1,3-glucanases. We also report high level resistance against Fusarium oxysporum in transgenic tomato plants expressing a specific combination of genes encoding these enzymes.

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Somaclonal variation on in vitro callus culture potato cultivars

Horticultura Brasileira ◽

10.1590/s0102-05362004000200027 ◽

2004 ◽

Vol 22 (2) ◽

pp. 300-304 ◽

Cited By ~ 28

Author(s):

Patricia N. Bordallo ◽

Derly H. Silva ◽

José Maria ◽

Cosme D. Cruz ◽

Elizabeth P. Fontes

Keyword(s):

Somaclonal Variation ◽

Culture Media ◽

Callus Formation ◽

Potato Cultivars ◽

Synthetic Seed ◽

Arbitrary Sequence ◽

Stem Explants ◽

Factors Affecting ◽

High Level

Synthetic seeds can be an alternative for those species in which botanical seeds are not viable. One of the major problems of in vitro plant cultivation is the high level of somaclonal variation. The most common factors affecting somaclonal variation are genotype, explant source, in vitro period and cultivation conditions in which the culture is established. In this work, calli were induced using leaf and stem explants of the commercial potato cultivars Achat, Baraka, Baronesa, Bintje, and Contenda in MS culture media supplemented with 1.65 mM of picloram and 11.5 mM of 2,4-D. Seventy and 90 days after induction, DNA samples of 40 calli were compared concerning the effects of the two explant (leaf and stem) and two growth regulator sources on five potatoes cultivars. A total of 20 arbitrary sequence primers were evaluated. The RAPD pattern generated by these primers suggested a high percentage of polymorphic fragments among the five genotypes, indicating a high level of genetic variation among cultivars. Cultivar Baronesa showed the highest number of polymorphic fragments for all treatments. The cultivar Contenda showed the smallest somaclonal variation, for most of the treatments, except for the treatment which consisted of stem explants, picloram (1.65 mM) application, and a 70-day period of callus formation. 'Contenda' is, therefore, the most suitable cultivar for synthetic seed production.

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Fluoroquinolone resistance in Clostridium difficile isolates from a prospective study of C. difficile infections in Europe

Journal of Medical Microbiology ◽

10.1099/jmm.0.47738-0 ◽

2008 ◽

Vol 57 (6) ◽

pp. 784-789 ◽

Cited By ~ 85

Author(s):

Patrizia Spigaglia ◽

Fabrizio Barbanti ◽

Paola Mastrantonio ◽

Jon S. Brazier ◽

Frédéric Barbut ◽

...

Keyword(s):

Amino Acid ◽

Clostridium Difficile ◽

Prospective Study ◽

Amino Acid Substitution ◽

Amino Acid Change ◽

Fluoroquinolone Resistance ◽

Genes Encoding ◽

High Level ◽

A Prospective Study

The European Study Group on Clostridium difficile (ESGCD) conducted a prospective study in 2005 to monitor and characterize C. difficile strains circulating in European hospitals, collecting 411 isolates. Eighty-three of these isolates, showing resistance or intermediate resistance to moxifloxacin (MX), were selected for this study to assess susceptibility to other fluoroquinolones (FQs) and to analyse the gyr genes, encoding the DNA gyrase subunits GyrA and GyrB. Twenty MX-susceptible isolates from the surveillance study were included for comparison. Overall, one amino acid substitution in GyrA (Thr82 to Ile) and four different substitutions in GyrB (Ser416 to Ala, Asp426 to Asn, Asp426 to Val and Arg447 to Lys) were identified. A high level of resistance (MIC ≥32 μg ml−1) to MX, ciprofloxacin (CI), gatifloxacin (GA) and levofloxacin (LE) was found in 68 isolates showing the amino acid substitution Thr82 to Ile in GyrA, in eight isolates with the substitutions Thr82 to Ile in GyrA and Ser416 to Ala in GyrB, in two isolates showing the substitution Asp426 to Asn in GyrB and in one isolate with Asp426 to Val in GyrB. The remaining four isolates showed high MICs for CI and LE, but different MIC levels for MX and GA. In particular, intermediate levels of resistance to MX were shown by two isolates, one with the substitution Thr82 to Ile in GyrA, and one showing Asp426 to Asn in GyrB. The substitution Arg447 to Lys in GyrB was found in two strains resistant to MX, CI and LE but susceptible to GA. No substitutions in GyrA were found in the FQ-susceptible strains, whereas two strains showed the amino acid change Ser416 to Ala in GyrB. Thr82 to Ile was the most frequent amino acid change identified in the C. difficile isolates examined. In contrast to previous observations, 10 % of the isolates showed this substitution in association with Ser416 to Ala in GyrB. The other amino acid changes found were characteristic of a few strains belonging to certain types and/or countries. Two new substitutions for C. difficile, Ser416 to Ala and Arg447 to Lys, were found in GyrB. Whereas the former does not seem to have a key role in resistance, since it was also detected in susceptible strains, the latter substitution occurred in the same position where other amino acid variations take place in resistant Escherichia coli and other C. difficile strains. A large number of C. difficile isolates now show an alarming pattern of resistance to the majority of FQs currently used in hospitals and outpatient settings, therefore judicious use of these antibiotics and continuous monitoring of in vitro resistance are necessary.

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The Genetic Engineering of Two Commercial Potato Cultivars for Resistance to Potato Virus X

Nature Biotechnology ◽

10.1038/nbt0389-273 ◽

1989 ◽

Vol 7 (3) ◽

pp. 273-278 ◽

Cited By ~ 58

Author(s):

André Hoekema ◽

Marianne J. Huisman ◽

Lucy Molendijk ◽

Peter J. M. van den Elzen ◽

Ben J. C. Cornelissen

Keyword(s):

Genetic Engineering ◽

Potato Virus ◽

Potato Virus X ◽

Potato Cultivars ◽

Commercial Potato

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Biochemical and Genetic Responses of Tea (Camellia sinensis (L.) Kuntze) Microplants under Mannitol-Induced Osmotic Stress In Vitro

Plants ◽

10.3390/plants9121795 ◽

2020 ◽

Vol 9 (12) ◽

pp. 1795

Author(s):

Lidiia Samarina ◽

Alexandra Matskiv ◽

Taisiya Simonyan ◽

Natalia Koninskaya ◽

Valentina Malyarovskaya ◽

...

Keyword(s):

Osmotic Stress ◽

Culture Media ◽

Epigallocatechin Gallate ◽

High Strength ◽

Growth And Yield ◽

Horticultural Crops ◽

Stress Related Genes ◽

High Level ◽

Genetic Responses

Osmotic stress is a major factor reducing the growth and yield of many horticultural crops worldwide. To reveal reliable markers of tolerant genotypes, we need a comprehensive understanding of the responsive mechanisms in crops. In vitro stress induction can be an efficient tool to study the mechanisms of responses in plants to help gain a better understanding of the physiological and genetic responses of plant tissues against each stress factor. In the present study, the osmotic stress was induced by addition of mannitol into the culture media to reveal biochemical and genetic responses of tea microplants. The contents of proline, threonine, epigallocatechin, and epigallocatechin gallate were increased in leaves during mannitol treatment. The expression level of several genes, namely DHN2, LOX1, LOX6, BAM, SUS1, TPS11, RS1, RS2, and SnRK1.3, was elevated by 2–10 times under mannitol-induced osmotic stress, while the expression of many other stress-related genes was not changed significantly. Surprisingly, down-regulation of the following genes, viz. bHLH12, bHLH7, bHLH21, bHLH43, CBF1, WRKY2, SWEET1, SWEET2, SWEET3, INV5, and LOX7, was observed. During this study, two major groups of highly correlated genes were observed. The first group included seven genes, namely CBF1, DHN3, HXK2,SnRK1.1, SPS, SWEET3, and SWEET1. The second group comprised eight genes, viz. DHN2, SnRK1.3, HXK3, RS1, RS2,LOX6, SUS4, and BAM5. A high level of correlation indicates the high strength connection of the genes which can be co-expressed or can be linked to the joint regulons. The present study demonstrates that tea plants develop several adaptations to cope under osmotic stress in vitro; however, some important stress-related genes were silent or downregulated in microplants.

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Genetically engineered resistance to potato virus X in four commercial potato cultivars

Plant Cell Reports ◽

10.1007/bf01690261 ◽

1995 ◽

Vol 15 (1-2) ◽

pp. 91-96 ◽

Cited By ~ 7

Author(s):

Huimin Xu ◽

Houri Khalilian ◽

Mohamed Eweida ◽

Sam Squire ◽

Mounir G. Abouhaidar

Keyword(s):

Potato Virus ◽

Potato Virus X ◽

Genetically Engineered ◽

Potato Cultivars ◽

Commercial Potato ◽

Engineered Resistance

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High Aspartyl Proteinase Production and Vaginitis in Human Immunodeficiency Virus-Infected Women

Journal of Clinical Microbiology ◽

10.1128/jcm.37.5.1376-1380.1999 ◽

1999 ◽

Vol 37 (5) ◽

pp. 1376-1380 ◽

Cited By ~ 30

Author(s):

F. de Bernardis ◽

F. Mondello ◽

G. Scaravelli ◽

A. Pachì ◽

A. Girolamo ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Fungal Resistance ◽

Curative Effect ◽

Proteinase Production ◽

Immunodeficiency Virus ◽

Low Levels ◽

High Level ◽

Pepstatin A

Vaginal isolates of Candida albicans from human immunodeficiency virus-positive (HIV+) and HIV− women with or without candidal vaginitis were examined for secretory aspartyl proteinase (Sap) production in vitro and in vivo and for the possible correlation of Sap production with pathology and antimycotic susceptibility in vitro. HIV+women with candidal vaginitis were infected by strains of C. albicans showing significantly higher levels of Sap, a virulence enzyme, than strains isolated from HIV+, C. albicans carrier subjects and HIV− subjects with vaginitis. The greater production of Sap in vitro was paralleled by greater amounts of Sap in the vaginal fluids of infected subjects. In an estrogen-dependent, rat vaginitis model, a strain of C. albicans producing a high level of Sap that was isolated from an HIV+ woman with vaginitis was more pathogenic than a strain of C. albicans that was isolated primarily from an HIV−, Candida carrier. In the same model, pepstatin A, a strong Sap inhibitor, exerted a strong curative effect on experimental vaginitis. No correlation was found between Sap production and antimycotic susceptibility, as most of the isolates were fully susceptible to fluconazole, itraconazole, and other antimycotics, regardless of their source (subjects infected with strains producing high or low levels of Sap, subjects with vaginitis or carrier subjects, or subjects with or without HIV). Thus, high Sap production is associated with virulence of C. albicans but not with fungal resistance to fluconazole in HIV-infected subjects, and Sap is a potentially new therapeutic target in candidal vaginitis.

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In vitro susceptibility of Clostridium difficile to rifaximin and rifampin in 359 consecutive isolates at a university hospital in Houston, Texas

Journal of Clinical Pathology ◽

10.1136/jcp.2009.071688 ◽

2010 ◽

Vol 63 (4) ◽

pp. 355-358 ◽

Cited By ~ 35

Author(s):

Z-D Jiang ◽

H L DuPont ◽

M La Rocco ◽

K W Garey

Keyword(s):

Clostridium Difficile ◽

In Vitro Study ◽

University Hospital ◽

Binary Toxin ◽

In Vitro Susceptibility ◽

Toxin Genes ◽

Genes Encoding ◽

Agar Dilution ◽

High Level

AimThis was an in vitro study to analyse the susceptibility of Clostridium difficile isolates to rifampin and rifaximin.MethodsStool samples from patients who had nosocomial diarrhoea and C difficile toxin B at a university hospital between August 2006 and December 2007 were cultured for C difficile. Susceptibility of C difficile isolates to rifaximin and rifampin was determined by agar dilution and E strips, respectively. C difficile isolates were analysed via PCR for genes encoding toxins A and B, for binary toxin (BT), and for partial deletions of the tcdC gene (tcdC-del).ResultsRifaximin exhibited high-level activity against 359 C difficile isolates, with MIC50 <0.01 μg/ml and MIC90 0.25 μg/ml; rifampin had MIC50 <0.002 μg/ml and MIC90 4 μg/ml. Among isolates analysed, 55 (15%) were positive for BT and tcdC-del. 28 (8% of 359) isolates were resistant to rifampin (≥32 μg/ml), of which 6 (2% of 359) were resistant to rifaximin and rifampin with MIC values ≥32 μg/ml. 2 of the 28 isolates resistant to rifampin were A+/B+/BT+/tcdC-del+, 5 were A+/B+/BT−/tcdC-del+, 4 were A+/B+/BT+/tcdC-del−, 13 were A+/B+/BT−/tcdC-del−, and 4 had no detectable toxin genes. Of the 11 isolates resistant to rifaximin alone, 1 was A+/B+/BT−/tcdC-del+, 2 were A+/B+/BT+/tcdC-del−, 6 were A+/B+/BT−/tcdC-del−, and 2 had no detectable toxin genes.ConclusionsThe study demonstrates that rifaximin has high-level activity against C difficile in vitro. Determination of resistance to rifampin by E strip did not predict rifaximin resistance.

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Genome-wide transposon screen of aPseudomonas syringae mexBmutant reveals the substrates of efflux transporters

10.1101/684605 ◽

2019 ◽

Cited By ~ 1

Author(s):

Tyler C. Helmann ◽

Caitlin L. Ongsarte ◽

Jennifer Lam ◽

Adam M. Deutschbauer ◽

Steven E. Lindow

Keyword(s):

Major Facilitator Superfamily ◽

Efflux Transporters ◽

Bacterial Strains ◽

Genome Wide ◽

Genes Encoding ◽

Resistance Nodulation Division ◽

Major Facilitator ◽

High Level ◽

Increased Susceptibility

AbstractBacteria express numerous efflux transporters that confer resistance to diverse toxicants present in their environment. Due to a high level of functional redundancy of these transporters, it is difficult to identify those that are of most importance in conferring resistance to specific compounds. The resistance-nodulation-division (RND) protein family is one such example of redundant transporters that are widespread among Gram-negative bacteria. Within this family, the MexAB-OprM protein complex is highly-expressed and conserved amongPseudomonasspecies. We exposed barcoded transposon mutant libraries in isogenic wild-type and ΔmexBbackgrounds inP. syringaeB728a to diverse toxic compoundsin vitroto identify mutants with increased susceptibility to these compounds. Mutants in genes encoding both known and novel redundant transporters, but with partially overlapping substrate specificities were observed in a ΔmexBbackground. Psyr_0228, an uncharacterized member of the Major Facilitator Superfamily of transporters, preferentially contributes to tolerance of acridine orange and acriflavine. Another transporter located in the inner membrane, Psyr_0541, contributes to tolerance to acriflavine and berberine. The presence of multiple redundant, genomically encoded, efflux transporters appears to enable bacterial strains to tolerate a diversity of environmental toxins. This genome-wide screen in a hyper-susceptible mutant strain revealed numerous transporters that would otherwise be dispensable in these conditions. Bacterial strains such asP. syringaethat likely encounter diverse toxins in their environment such as in association with many different plant species, probably benefit from possessing multiple redundant transporters that enable versatility to tolerate novel toxicants.

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Characterization of Mycoplasma hominis mutations involved in resistance to fluoroquinolones.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.41.2.269 ◽

1997 ◽

Vol 41 (2) ◽

pp. 269-273 ◽

Cited By ~ 37

Author(s):

C M Bebear ◽

J M Bové ◽

C Bebear ◽

J Renaudin

Keyword(s):

Reference Strain ◽

Bacterial Species ◽

Mycoplasma Hominis ◽

Base Change ◽

Fluoroquinolone Resistance ◽

Genes Encoding ◽

One Step ◽

Resistant Mutants ◽

High Level

Fluoroquinolone-resistant mutants of Mycoplasma hominis were selected in vitro from the PG21 susceptible reference strain either by multistep selection on increasing concentrations of various fluoroquinolones or by one-step selection on agar medium with ofloxacin. The quinolone resistance-determining regions (QRDR) of the structural genes encoding the A and b subunits of DNA gyrase were amplified by PCR, and the nucleotide sequences of eight multistep-selected resistant strains were compared to those of susceptible strain PG21. Four high-level resistant mutants that were selected on norfloxacin or ofloxacin contained a C-to-T transition in the gyrA QRDR, leading to substitution of Ser-83 by Leu in the GyrA protein. Analysis of the sequence of the gyrB QRDR of the eight multistep-selected mutants did not reveal any difference compared to that of the gyrB QRDR of the reference strain M. hominis PG21. Similar analyses of eight one-step-selected mutants did not reveal any base change in the gyrA and gyrB QRDRs. These results suggest that in M. hominis, like in other bacterial species, a gyrA mutation at Ser-83 is associated with fluoroquinolone resistance.

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