GCP Project G7009.07: Cloning, characterization and

validation of AltSB/Al tolerance in rice

PI: Leon Kochian, USDA-ARS, Cornell University Co-PI: Susan McCouch, Department of Plant Breeding, Cornell University

Comparative Genomics Challenge Initiative • Use discovery of sorghum Al tolerance gene, SbMATE, to identify rice Al tolerance genes for improving rice production on acid soils. • Found out early in this project that rice, the most Al tolerant cereal, doesn’t use the root tip Al exclusion mechanism based on Al-activated organic acid release as a major Al tolerance mechanism as in sorghum, wheat, barley, rye and to a degree, maize.

Al Exclusion Via Organic Acid Release

• Rice truly tolerates Al accumulated in root tip cell wall and symplasm. • No correlation between root tip citrate/malate release and Al tolerance. • Ma’s lab has cloned several candidate rice Al tolerance genes involved in Al uptake, cell wall modification, and ART1 which may regulate expression of other tolerance genes

Ma (2007) Syndrome of Al toxicity & diversity of Al resistance in higher plants. Int. Rev. Cyt. 264:225-253

Candidate Rice Al Tolerance Genes Identified via Forward Genetics Approaches

• Art1: Al regulated transcription factor that activates the expression of candidate tolerance genes. • STAR1/2: PM ABC transporter that exports UDP-Glucose to the cell wall, possibly reducing Al binding. • FRL4: MATE citrate transporter (homolog of SbMATE) that plays a minor role in tolerance. • Nrat1 & ALS1: Al transporters that absorb Al from cell wall (Nrat1) and sequester it in vacuole. • Ma found that none of these genes mapped to previously identified Al tolerance QTL

Conducted Joint Linkage-Genome Wide Association Analysis of Rice Al Tolerance

Trait Index Chr QTL Flanking Markers LOD Allele effect R2

TRG 1 Alttrg1.1 RM319/RM315 4.56 Azucena 0.095

TRG 2 Alttrg2.1 RM526/RM318 3.44 Ir64 0.059

LRG 9 Altlrg9.1 RM257/RM160 6.57 Azucena 0.165

TRG 12 Alttrg12.1 RM453/RM512 7.85 Azucena 0.193

QTL Analysis of IR64 x Azucena RIL population (Azucena is Al tolerant trop japonica and Ir64 is Al sensitive indica)

• Conducted high resolution mapping of largest QTL on chr 12. • Gene responsible for chr 12 QTL is ART1, the zinc-finger transcription factor that activates the expression of candidate rice Al tolerance genes in response to Al. • Generating NILs with tolerant ART1 allele (Azucena) introgressed into Al sensitive IR64 and also reciprocal NIL. • Valuable breeding line for improving indica Al tolerance and to study effect of different ART1 alleles on expression of other tolerance genes • Also studying ART1 allelic diversity in rice association panel to identify best alleles. Evaluating phenotypic impact via rice transformation

GWAS of Aluminum Tolerance

All /EMMA

indica

aus

trop jap

temp jap

MATE Efflux protein

MATE Efflux protein

Root elongation

STAR2

Isocitrate lyase

Isocitrate lyase

Nrat candidate gene 8 SNPs; p=2.8E-07

Ferriportin domain protein

Iron-sulfur binding Magnesium-transporter

ART1

C2H2 TF

Outlier Analysis

1 Million SNP Chip

Generation of Near Isogenic Lines for QTL on Chr 1, 2, 9 and 12

• Will have NILs for each of these QTL in both IR64 and Azucena backgrounds by mid 2014.

• Am pyramiding the superior alleles for the 4 QTL (3 from Azucena and 1 from IR64) in IR64.

• Hope to also pyramid these QTL with superior OsPSTOL1 (Pup1) allele

• Breeding lines for increasing rice Al tolerance and acid soil tolerance.

• Experimental materials for identifying novel Al tolerance mechanisms/genes (chr 1,2, and 9)

GWAS Analysis of the PM Al Transporter: Nrat1

GWAS Peak on chr 2 colocalizes with Nrat1 and explains 40% of the variation in aus Al tolerance.

Zoom-in view of GWAS peak co-localizing with Nrat1 in aus. Most significant SNP is marked by an asterisk. Nrat1 is indicated by a red circle. Dr. Jianyong Li

**

**

*

Sensitive Aus

Tolerant Aus Indica

Japonica

Region Promoter Coding sequence SNPs M2 M3 M4 M5 M6 M8 M9 M12 M13 M15

Location(MSU V7) 2661026 2661163 2661328 2661476 2661525 1662980 1663056 1663674 1664197 1664242 Sensitive aus G G T C G T A A T C

Tolerant aus, indica and japonica

A T A T T C G G C T

Tolerant and Sensitive Nrat1 Haplotypes Due to SNPs in both Promoter and Coding Regions

Al tolerance of aus lines harboring tolerant and sensitive Nrat1 haplotypes.

SNPs in tolerant allele associated both with increased Nrat1 expression and higher Al uptake

Al-Induced Nrat1 Expression

Functional characterization of Nrat1 in yeast. A: time course of yeast growth in Al (50 µM). B: Al uptake in yeast cells exposed to 50 to 200 µM Al. Black symbols/bars: cells expressing the empty vector (control); white symbols/ bars: cells expressing tolerant Nrat1-1; gray symbols/bars: cells expressing sensitive Nrat1-2.

CONTROL

150 µM Al

Col 2a 2b 1a 1b

Root growth of representative plants from four independent transgenic lines over-expressing Nrat1, grown in the absence (top) or presence (bottom) of Al. Col, wild-type Columbia; 2a, 2b: lines expressing sensitive Nrat1-2; 1a, 1b, lines expressing tolerant Nrat1-1.

Functional Analysis of Tolerant and Sensitive Nrat1 Alleles in Yeast and Transgenic Arabidopsis

Hap1: Tol. Allele Hap2: Sens. Allele

Sensitive Allele

Tolerant Allele

• Joint linkage-GWA analysis of rice Al tolerance has validated candidate genes as bona fide Al tolerance genes (ART1 and Nrat1).

• Linkage analysis identified ART1 and GWA analysis identified Nrat1.

• This analysis has also identified a number of novel candidate Al tolerance regions possibly involved in cell wall tolerance and Al transport.

• Variation in Nrat1 DNA sequence results in tolerant and sensitive versions of this PM Al uptake transporter. The tolerant Nrat1 transporter mediates greater Al influx across the root cell PM, resulting in lower Al levels in the cell wall.

• Al uptake via Nrat1 appears to be a major Al tolerance mechanism. There may be functional Nrat1 orthologs in maize (work by Claudia Guimaraes) and sorghum.

Summary

PRODUCTS • Markers flanking Al tolerance QTL on rice chr 1,2 , 9 and 12 • NILs harboring tolerant alleles of each of the 4 QTL regions in Al both IR64 and Azucena background s as well as all 4 QTL will be completed in 2014 to use for breeding for improved Al tolerance. • NILs harboring QTL on chr 1,2 and 9 a resource for identifying rice novel Al tolerance genes and mechanisms. • Sequence for tolerant and sensitive alleles of two major Al tolerance genes, ART1 and Nrat1. • Transgenic wheat and maize expressing the tolerant Nrat1 allele.