Research

Overview

Research in the Wise laboratory is focused on the high-throughput functional analysis of important agronomic genes in cereal crops. We use a variety of interdisciplinary approaches, including plant and microbial genetics, molecular biology, plant pathology, and bioinformatics & computational biology. 

THE SYSTEMS WE WORK ON . . .

Host-pathogen interactions:

Plant pathogens are among the greatest threats to crop production worldwide, resulting in yield losses of 10-20% each year. Obligate biotrophic fungi (e.g., mildews and rusts) require a living host to survive and cause some of the most destructive epidemics. Because they are unable to survive autonomously, obligate parasites also represent ideal tools for exploring interdependent signaling between disease agents and their hosts. Pathogens secrete effectors to modulate immune responses, primary and secondary metabolism, and other cellular processes to enable colonization of the host. Many aspects of defense have been explored using a typical gene-by-gene approach, yet the dynamics of initiation, and the temporal and spatial control of these immune processes are not well understood.

To tackle this challenge, we are exploiting the biotrophic interaction between the powdery mildew fungus, Blumeria graminis f. sp. hordei (Bgh), and its diploid host plant, barley (Hordeum vulgare L.) (Fig 1). In this system, the outcome is determined largely by the plant’s response to secreted effectors. Disease is blocked by host immune receptors encoded by resistance (R) genes, designated Ml (for mildew resistance). Both the 5.1-Gb barley and 130-Mb Bgh genomes have high-quality reference sequences, and we are using these resources to interrogate key aspects of cereal immunity.

THE OVER-ARCHING QUESTIONS WE SEEK TO ADDRESS ARE:

Text Box:    Fig 1. Key interaction stages of Bgh with its host plant, barley. A) Penetration by appressorial germ tube at 16 hours after inoculation (HAI). B) Haustorial feeding structure inside epidermal cell at 32 – 48 HAI. C) 5-7 days; sporulating conidiophores that re-initiate the (asexual) infection cycle.  Micrographs by Tim Carver.
Fig 1. Key interaction stages of Bgh with its host plant, barley. A) Penetration by appressorial germ tube at 16 hours after inoculation (HAI). B) Haustorial feeding structure inside epidermal cell at 32 – 48 HAI. C) 5-7 days; sporulating conidiophores that re-initiate the (asexual) infection cycle. Micrographs by Tim Carver.

Immune signaling mutants and ‘omic-level’ data sets for system-wide integration:

To set the stage for our investigations, we isolated several fast-neutron-mutants from barley line CI 16151 (harboring the Mla6 R gene) and backcrossed to achieve homozygosity. Shown in Fig 2: A) Mla6 recognizes AVRa6 effector from Bgh isolate 5874; plants with this allele are resistant. B) Blufensin1 (Bln1) is a negative regulator of PTI signaling (Meng et al. 2009) and the bln1 mutant exhibits enhanced basal defense (Xu et al. 2015). Overexpression of Bln1 or its unlinked family member, Bln2, increases susceptibility to Bgh in compatible interactions, while Barley stripe mosaic virus (BSMV) Induced Gene Silencing (VIGS) increases resistance (Xu et al. 2015). C) The mla6 + bln1 double mutant is susceptible (due to the mla6 deletion), but PTI-related cellular pathways are deregulated in this background. D) mla6 mutants are susceptible (Moscou et al. 2011a). E) Rar3 is a novel locus required for Mla6 function, but segregates independently of both Mla6 and Rar1.

The panel shown in Fig 2 was used to generate ‘omic’ level datasets for host & pathogen at key stages of Bgh infection: appressorium formation, penetration of epidermal cells, and development of haustorial feeding structures. Bgh-inoculated 1st leaves (5 genotypes x 6 time points x 3 biological replications) were sampled from a split-plot design at 0, 16, 20, 24, 32, and 48 hours after inoculation (HAI) and used as substrate for 1) Illumina RNA-Seq, 2) Illumina small RNA-Seq, 3) PacBio Iso-Seq (62,578 full-length transcripts), and 4) construction of a 3-frame yeast two-hybrid prey library for next-generation interaction screening with 100 select Bgh effectors as baits.

Text Box:    Fig 1. Key interaction stages of Bgh with its host plant, barley. A) Penetration by appressorial germ tube at 16 hours after inoculation (HAI). B) Haustorial feeding structure inside epidermal cell at 32 – 48 HAI. C) 5-7 days; sporulating conidiophores that re-initiate the (asexual) infection cycle.  Micrographs by Tim Carver.

Fig 2. Bgh-infected, barley CI 16151 immune mutants.

 

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