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.

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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.

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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

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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

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Medicago truncatula

Switchgrass

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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

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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

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irg1

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

Wild-type R108

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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

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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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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)