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Biotechnological approaches to improve switchgrass . Kiran Mysore... · PDF file • Biomass yield is a highly complex trait • miRNAs have emerged as a prominent class of gene regulatory

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Text of Biotechnological approaches to improve switchgrass . Kiran Mysore... · PDF file •...

  • Kiran Mysore (Noble) Srinivasa Rao Uppalapati (Noble) Upinder Gill (Noble) Chunxiang Fu (Noble) Ramanjulu Sunkar (OSU) Zeng-Yu Wang (Noble)

    Biotechnological approaches to improve switchgrass production

  • • Biomass yield is a highly complex trait

    • miRNAs have emerged as a prominent class of gene regulatory factors

    • Plant miR156 is a family of small, non-coding, endogenous RNAs with a relatively high expression level in the juvenile phase of plants

    • most members of the SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) transcription factor family are targeted by miR156 in plants

    Use of micro RNA (miRNA) for plant biomass improvement

  • (a) Representative plants from each group are shown. (b) The miR156 level of transgenic plants detected and quantified by quantitative real-time PCR. (c) Small RNA blot analysis of miR156 level in transgenic plants.

    Ctrl T-35 T-37 T-44 R

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    Group I Group II Group III

    miR156

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    T-14 T-35 T-40 T-27 T-32 T-44 T-34 T-37 Ctrl

    (a)

    (c)

    Group I Group II Group III Group I Group II Group III

    Morphological and molecular characterization of transgenic switchgrass plants overexpressing miR156b

  • Tiller number (a) and biomass yield (b) of transgenic switchgrass plants

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  • SPL: SQUAMOSA PROMOTER BINDING PROTEIN LIKE Transcript abundance of PvSPL1, PvSPL2, PvSPL3, and PvSPL6 was revealed by quantitative RT-PCR.

    (a)

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    Group I Group II Group III

    (b)

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    0.0000

    0.0012

    0.0024

    0.0036

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    0.0060 PvSPL3 PvSPL6

    Group I Group II Group III

    Transcript abundance of putative miR156-targeted SPL genes in transgenic switchgrass plants.

  • Overexpression of miR156b inhibits inflorescence development but not the in vitro propagation

    Control Group II Group II TmiR156OE-92 Control

  • Diseases could negatively impact switchgrass biomass. Genetic vulnerability to diseases is a very serious concern when one switchgrass cultivar is grown in monoculture.

    Leaf and stem rust: A growing concern

    Puccinia emaculata is identified as the major rust pathogen of switchgrass

  • Specific Objectives

    Identify sources of host resistance in switchgrass against P. emaculata Identify genetic variations for rust resistance in various switchgrass populations (cultivars). Identify genes involved in nonhost resistance (NHR) to P. emaculata “NHR is defined as a form resistance exhibited by entire plant species to a particular microbial pathogen, which is the most common and durable form of resistance”- Heath, 2000. Identify economically viable chemical control for switchgrass rust Fungicides can be very expensive and will increase the cost of production. It is important to identify alternative chemical sources to control switchgrass rust that will be economically feasible.

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    Growth chamber evaluations Field evaluations

    Score classes Score classes

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    Severity of rust infection in various switchgrass cultivars

    Uppalapati et al., (2013) BioEnergy Res. DOI 10.1007/s12155-012-9263-6

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    Mean rust scores in various switchgrass cultivars

    Uppalapati et al., (2013) BioEnergy Res. DOI 10.1007/s12155-012-9263-6

  • Mapping populations and RNAseq

    Challenge with rust and isolate RNA at 0, 12 and 60 hpi

    Whole genome RNA-seq for candidate gene and SNP discovery is in progress

  • Initial characterization of nonhost interactions

    Mellersh and Heath, 2003

    LG LG

    Medicago truncatula

    Switchgrass

    Host Plant

    Nonhost Plant

  • Development of a Medicago Tnt1-insertion population for forward and reverse genetics

    ● Tnt1 is a retrotransposon: transposition is replicative and insertions are random and stable

    ● Tnt1 is activated by somatic embryogenesis

    ● An R108 starter line containing 5 Tnt1 insertions was used to generate the Tnt1 mutant population via tissue culture

    LTR retrotransposon Copy / Paste

    mRNA Copy

    insertion

    A B

    C D

    E

    Tadege et al., 2008, Plant Journal., 54:335-347 Tadege et al., 2005, Trends Plant Sci., 10:229-235

    Tadege et al., 2009, Plant Physiol., 151:978-984

  • To date, ~2000 Tnt1 lines are screened

    Spray inoculation with Pe spores (1 X 105 spores/ml; 0.001% Tween 20); 15 ml per tray)

    Enhanced susceptibility or resistance phenotype

    Dew chamber-24 h (19 °C; 24D) and further incubation in growth chamber (22°C/19°C; 12L:12D) for 7-10 days

    Characterization of interesting mutants

    12 plants/Tnt1 line (23 lines/week)

    Tnt1-insertional mutant screening in M. truncatula

    1. ROS localization (2 dpi) 2. WGA staining – initial events (3 dpi) 3. Aniline blue staining for CWA (5 dpi ) 4. Macroscopic symptoms (14 dpi)

  • M. truncatula Tnt1 insertion mutants with various phenotypes

    Wild-type (R108; Long germ tube and no penetration

    No spore germination Accumulation of phenolics around the spore

    Accumulation of phenolics around germinated spore

    Very short germtube Short germtube

    Attempted penetration with plant cell death

    Attempted penetration through stomata with guard

    cell death

    Attempted penetration through stomata without

    cell death

  • irg1

    Tnt1-insertional mutant screening in M. truncatula identified inhibitor of rust germ-tube differentiation1 (irg1)

    Wild-type R108

    Pe rc

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    Uppalapati et al., (2012) Plant Cell, 24:353-370

    Only the abaxial leaf surfaces inhibit rust germ-tube differentiation on irg1 plants

  • IRG1 encoded a Cys(2)His(2) zinc finger transcription factor (PALM1) that also controls dissected leaf morphology in

    Medicago truncatula

  • Multiple mutant alleles of IRG1/PALM1 inhibit rust germ-tube differentiation

    Uppalapati et al., (2012) Plant Cell, 24:353-370

  • irg1/palm1 mutation results in loss of abaxial epicuticular wax loading

    Uppalapati et al., (2012) Plant Cell, 24:353-370

  • Overexpression of LTP or CER2 may result in asymmetric loading of epicuticular waxes in irg1

    Samuels et al., 2008

  • Response of various monocot plants to switchgrass rust pathogen inoculation

    O O O O O O O A A A A A A A H H H H H H H S S S S S S S

    Switchgrass

    O = Oriented growth A = Appressoria H = Haustoria S = Sporulation

    Uppalapati et al., (2013) in pre