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Title: HOPX1 Ea (Eop3) in Erwinia Amylovora functions as an avirulence gene in apple and is regulated by HRPL

Author
item BOCSANCZY, ANA MARIA - Cornell University
item Schneider, David
item DECLERCK, GENEVIEVE - Cornell University
item Cartinhour, Samuel
item BEER, STEVEN - Cornell University

Submitted to: Journal of Bacteriology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/14/2011
Publication Date: 11/28/2011
Citation: Bocsanczy, A., Schneider, D.J., Declerck, G.A., Cartinhour, S.W., Beer, S.V. 2011. HOPX1 Ea (Eop3) in Erwinia Amylovora functions as an avirulence gene in apple and is regulated by HRPL. Journal of Bacteriology. 194:553-560.

Interpretive Summary: Fire blight is a devastating bacterial disease of apples, pears and related plants that is caused by the bacterium Erwinia amylovora. This pathogen delivers virulence proteins directly into host cells by means of a special system present in many pathogenic bacteria. Expression of this system and associated delivered proteins, called effectors, is controlled by a protein that binds to segments of DNA known as “hrp boxes”, which are located “upstream” of the regulated genes. Several models were developed by laboratory and computational efforts that identified putative hrp boxes in the DNA sequence of a highly virulent strain of E. amylovora. Downstream genes, including candidate effectors and virulence factors, were identified based on similarity to known factors of other pathogenic bacteria. Deletion mutants of two candidates downstream of functionally confirmed hrp boxes were generated. These mutants did not differ in virulence (disease causing ability) from the wild-type strain when assayed in pear fruit and apple shoots. However, a mutant that over-expressed one of the candidate proteins suppressed the development of lesion formation in Nicotiana benthamiana, a frequently used experimental plant. In addition, the over-expressing mutant caused greater lesion formation in N. tabacum, which is indicative of the plant immune response. It also significantly reduced the progress of disease in apple shoots. These observations suggest that this bacterial protein is recognized by the innate immune system in apple shoots.

Technical Abstract: Fire blight is a devastating disease of rosaceous plants caused by the Gram-negative bacterium E. amylovora. This pathogen delivers virulence proteins into host cells utilizing the Type-Three Secretion System (T3SS). Expression of the T3SS and associated substrates are activated by the alternative sigma factor HrpL, which recognizes hrp box promoters upstream of regulated genes. A collection of hidden Markov models (HMM), developed by iterating between laboratory and computational approaches, was used to identify putative hrp boxes in the genome sequence of Ea273, a highly virulent strain of E. amylovora. Candidate effectors and virulence factors in downstream operons were identified based on homologies to known factors of other pathogenic bacteria. This set included proteins involved in flagella biosynthesis and those related to virulence functions of animal and plant pathogens. Deletion mutants of two candidates downstream of functionally confirmed hrp boxes, hopX1Ea and hopAK1Ea, were generated. These mutants did not differ in virulence from the wild-type strain when assayed in pear fruit and apple shoots. A mutant of Ea273 over-expressing hopX1Ea suppressed the development of HR when inoculated to Nicotiana benthamiana; however it contributed to HR in N. tabacum and significantly reduced the progress of disease in apple shoots, suggesting that HopX1Ea may act as an avirulence protein in apple shoots.