"Objective 1. Detailed annotation and reconstruction of NLR-ID genes and gene pairs in Oryza species.
Comparative analysis of cereal immune receptor uncovers that 19 pairs of distinct rice NLR/NLR-ID, only present in rice, whose ID could overlap with potential effector targets of all cereals simultaneously. Cereal crops in the Poaceae family are adapted by the different Puccinia spp. Rice - a distant relative of cereal crops species including wheat, barley, maize and sorghum – does not have its own adapted rust pathogens. Based on cell biology, haustorial development was observed within rice mesophyll cells, however sporulation was not observed on any rice accession, which suggest that intracellular receptor is involved in the resistance of rice to cereal rust. The phylogeny of cereals showed that cereals descended from the common ancestor, and divergence event of rice occurred earlier than wheat and barley but later than maize and sorghum, which indicate that rice has its own distinct immune components. To test if rice NLR/NLR-ID pairs might confer non-host resistance to cereal rust, comparative analysis of cereal immune receptors was carried out based on genome sequences of maize, sorghum, wheat, barley and all Oryza species. Furthermore, overlapping event of ID and potential effector target were defined by phylogeny including all the sequences of ID homology and ID. The results showed that 19 pairs of rice NLR/NLR-ID are likely candidates as sources of ""non-host"" disease resistance, and which could be introduced into barley Golden Promise.
Objective 2. Determine whether rice IDs in NLR-ID genes interact with wheat pathogen effectors.
Yeast 2-hybrid cDNA library was not performed, because our US collaborator showed evidence that effectors identified based on IDs of NLR-ID might not be recognized by paired NLR/NLR-ID. Instead, core effectors of cereal rusts were identified, synthesized and set up for testing for recognition by rice. All together phenotypes of cereal rust in rice suggest that core components of cereal rusts are recognized by rice. Comparative analysis of the secretomes of ten rust species, five being different races of Puccinia striiformis f. sp. tritici, uncovered 192 core effectors of cereal rusts. 98 of these 192 core effectors were synthesized and cloned. These effectors under the control of 35S promoter, assembled using GoldenGate methods, were used for assay of NLR/NLR-ID perception in transient assays in N. benthamiana. Moreover, for test of recognition phenotypes of each effector in rice, two independent systems were used to deliver these effectors into rice. Pseudomonas syringae pv. oryzae strain engineered were used to deliver each of these effectors, by type III secretion of AvrRps4. Foxtail mosaic virus vectors engineered will be also used to deliver each of these effectors.
Objective 3. Transform rice NLR/NLR-ID pairs identified in objective 2 into barley, and assay non-host resistance mediated by these rice NLR-IDs to rusts and other pathogens.
To address whether heterologous expression of paired rice NLR-IDs work in barley, under the control of RGA4/RGA5’s divergent promoters or RPR1/RPR2’s divergent promoters, RPR1/RPR2, RGA4/RGA5, GFP/GUS were introduced into barley respectively. As tested in T0 transgenic barley plants, RGA4/RGA5 promoter could function. All T1 seeds were harvested on the 80 lines of T0 plants and further resistance test will be done in T2 plants. For the test of resistance phenotypes, Pseudomonas syringae pv. oryzae strain engineered were used to deliver each of AVR1-CO39 or AVR-Pia, by type III secretion of AvrRps4 or AvrRpm1, and foxtail mosaic virus engineered were also used to deliver each of both effectors. As for Objective 2, it was necessary to adjust my intended plan which was to transform rice NLR/NLR-ID pairs identified in objective 2 into barley. Instead, of 19 pairs of NLR/NLR-ID identified in objective 1, I transformed 13 pairs of them into barley (Golden Promise) respectively. All T1 seeds were harvested on the 172 lines of T0 plants and further resistance test will be done in T2 plants. Two independent systems were used to deliver effectors into transgenic barley for phenotype test. Pseudomonas syringae pv. oryzae strain engineered were used to deliver each of AVR1-CO39, AVR-Pia or rust core effectors, by type III secretion of AvrRps4, and foxtail mosaic virus engineered will be also used to deliver each of these effectors."
