biotechnological approaches to improve switchgrass production. kiran mysore... · • biomass yield...
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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
U6
T-14 T-35 T-40 T-27 T-32 T-44 T-34 T-37 Ctrl
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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
Tille
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Group I Group II Group III D
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SPL: SQUAMOSA PROMOTER BINDING PROTEIN LIKE Transcript abundance of PvSPL1, PvSPL2, PvSPL3, and PvSPL6 was revealed by quantitative RT-PCR.
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0.007
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0.035 PvSPL1 PvSPL2
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0.0000
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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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Cave-in Rock
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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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Lowland Upland
Field Growth chamber
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Alamo Kanlow CIR Summer
Field Growth chamber a b
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Kanlow Alamo
Cave-in-Rock
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
Perc
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Ge Gt Ap Pn Ge Gt Ap Pn 0
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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 preparation
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Brachypodium as a monocot nonhost model system for the switchgrass rust pathogen
Mesophyll cell death?
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3-1 18-1 21 21-3 29-1 30-1
%U
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Germination Oriented growth Appressoria Infection foci
7 dpi, n=50-75
Uppalapati et al., (2013) in preparation
Four weeks after transplanting
Spray with P. emaculata
Spray with Switchgrass rust
24 lines (12 plants per line)
WGA staining 72 hai
Germination, Oriented growth, Appressoria and HR
Symptoms 14dpi
Screening of T-DNA insertion lines
400 lines tested so far
Upinder Gill, Unpublished
Phosphite-Induced priming in Arabidopsis infected with Hyaloperonospora arabidopsidis
Plant Physiol. 159:286-298, 2012
A) distorted hyphae, abundant hyphal swellings and short, stunted branches B) lysis of hyphal wall and release of cytoplasmic contents
ICPP 2008 9th International Congress of Plant Pathology, 24 - 29 August, Torino, Italy
Phi inhibits oomycetes growth in vitro
Role of Phosphite (Phi) in plant defense
Affect of Phi on switchgrass rust
100 mM Phosphite 0 mM Phosphite
Control 100mM Phi_24hr 100mM Phi_24hr_washed
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G= Germination O= Oriented growth A= Appressoria
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AOS LOX1 OPR3 CHI CHS1 PAL1 Aox1A ChiA PR10 PR5
Mock phi_1
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Phi Induces chitinases and phenylpropanoid pathway genes in switchgrass
Upinder Gill, Unpublished
• Overexpression of miR156 in switchgrass significantly increased the plant biomass.
• Identified the major switchgrass rust pathogen in Oklahoma as Puccinia emaculata and determined the genetic diversity of rust resistance in upland and lowland cultivars of switchgrass.
• Characterized the interaction of P. emaculata on nonhost plant Medicago truncatula and established forward genetics screen using Tnt1 mutants to identify novel sources of resistance.
• Identified IRGI/PALM1 that plays role in asymmetric wax loading on
leaf surface that acts as a surface signal for pre-infection structure formation of P. emaculata.
• Established B. distachyon as a monocot model to study nonhost resistance against P. emaculata.
• Spraying phosphite on switchgrass can control rust disease.
Summary
Yongfeng Zhang Srinivasa Rao Uppalapati
Collaborators: Malay Saha (Noble) Steve Marek (OSU)
Acknowledgements
Yasuhiro Ishiga Shipra Mittal Upinder Gill
Chunxian Fu Zeng-Yu Wang Ramanjulu Sunkar (OSU)