Project Number: 6028-21000-011-34-R
Project Type: Reimbursable Cooperative Agreement
Start Date: Mar 1, 2020
End Date: Feb 28, 2023
Our long-term goal is to determine how weedy rice achieves competitiveness, and identify loci that can be exploited for weedy control or crop improvement. We will do this by characterizing three key features of weedy rice growth and reproduction that allow it to so aggressively outcompete its domesticated relatives: altered root system architecture (determining below-ground resource acquisition), highly shattering seed (conferring differential reproductive success and dispersal ability), and differential pathogen resistance (permitting a competitive advantage in U.S. crop fields). Building on our 13-year collaboration in Plant Genome Research Program (PGRP)-funded weedy rice research, this project will address the following specific aims: 1. Determine the genetic basis of the root system architecture (RSA) that has evolved convergently with repeated origins of weedy rice, and establish the connection between weedy rice RSA and weed competition via differential nutrient uptake. 2. Identify the genetic basis of seed shattering in independently evolved populations of weedy rice, and establish the mechanisms underlying the evolutionary lability of this trait. 3. Determine the genetic and biochemical bases underlying the differential resilience of U.S. weedy rice to rice blast disease, a major fungal pathogen of rice (Jia, ARS). Three subaims are: 1) What is the genetic basis of blast resistance in independently-evolved weed strains? 2) What is the effector recognized by PtrBHA in black hulled awn weedy rice? 3) What other plant and pathogen components are involved in PtrBHA mediated disease resistance? Approaches to address these three subaims are described in the following section.
Advance two new weed x crop mapping populations and identify blast resistance quantitative trail loci (QTLs) under greenhouse conditions. We will genotype of each recombinant inbred line (RIL) at F5 with Genotyping by sequencing. The genotyped RILs will be used in other aims, besides forming the basis of our examination of blast resistance in independently evolved weed rice. Reactions to 11 common blast races will be evaluated in parents and RILs with a complete randomized block design under greenhouse conditions. Patterns of blast resistance efficacies will be compared with an assembly of domesticated cultivars, to determine the degree to which different weedy rice lineages are competitively adapted to resisting blast fungus. The positions of mapped QTL, as well as overlap with known blast resistance genes, will be informative to the degree to which weedy rice blast resistance involves shared and/or novel loci. The candidate genes underlying major resistance QTL will be identified and a few of them will be analyzed in broader germplasm collections to understand genetic mechanism of their adaptation. To identify avirulence (AVR)- PtrBHA we will perform deep sequencing (20X=1MB) of the 20 differential blast races. The presence and absence of predicted AVR genes in sequenced genome will be used to correlate with disease reactions of wild type Katy and Mutant M2354. The absence of the predicted AVR genes in virulent differential blast races must correlate the presence of putative AVR genes in avirulent blast races. Because the strain IB-33 has AVR -PtrBHA, the absences of orthologous genomic sequences of any predicted AVR genes in other sequenced races in compared with IB-33 will help to delimit the genomic region harboring AVR- PtrBHA. Subsequent data analysis will be performed using Jump Genomics 9. The presence and absence of AVR genes will be verified by polymerase chain reaction (PCR) and analyzed with JMP14. A complementation test will be performed to validate the function of AVR- PtrBHA. To determine additional components involved blast resistance, the yeast two hybrid (Y2H) screening and interaction will be used. Candidate AVR genes will be examined in Y2H with Pi-ta and Ptr. To ensure success of Y2H screening different domains of PtrBHA will be used to make in frame fusion proteins with GAL4 DNA domain to screen positive interactors. The positive interactors will be identified from Y2H and verified using in vitro protein pull-down and in vivo biomolecular fluorescence complementation. We will then examine interactions of different domains of PtrBHA with selected interactors to identify critical domains/amino acid residues. We will then examine expression of these interacting genes in incompatible interactions (resistant, white rice Katy with IB49 versus weedy black hulled awn rice with (IB33), and in a compatible interaction (susceptible, S=Katy with IB-33) (3 biological replicates) at different time points post inoculation by semi quantitative and real time reverse transcription (RT)- polymerase chain reaction (PCR) to define weedy rice specific genetic and biochemical pathways leading to resistance to Magnaporthe. oryzae.