Proteomics Based Study of Soybean and Phakopsora pachyrhizi Interaction

Phakopsora pachyrhizi, the causal agent of Asian soybean rust (ASR), has the potential to cause severe yield losses as all currently grown U.S. commercial soybean varieties are susceptible. In this proteomics study, we compared two soybean sibling lines, a resistant line 10G18 and a susceptible line 10G21 derived from Recombinant Inbred Line (RIL) population RN06-32-2 to understand the compatible and incompatible host-pathogen interactions at the molecular level. We compared the protein profile differences between the two lines over a time-course of 14 days with or without P. pachyrhizi inoculation using differential in-gel electrophoresis (DIGE). Approximately 70 differentially expressed spots between 10G18 and 10G21 lines with and without P. pachyrhizi inoculation were identified. Some of these spots, which were upand down-regulated in resistant line 10G18, were sequenced using LC-MS/MS. Of the 70 differentially expressed protein spots, 31 up-regulated and 6 down-regulated spots in resistant line 10G18 were sequenced. These sequenced proteins were mostly involved in photosynthesis based on homology searches. The involvement in disease resistance for some of these differentially up-regulated proteins has been reported, indicating their possible role in soybean defense against ASR. However, further studies are necessary. Introduction Asian soybean rust (ASR) caused by Phakopsora pachyrhizi was first observed in Japan in 1902 and has since spread throughout the world (24). For several decades, the pathogen was slowly spreading in East Asia and Australia (10,30,31). ASR was first reported in Africa in late 1990s and in South America in 2001 (42). In November 2004, P. pachyrhizi reached North America. It was reported in Louisiana and several other southeastern states (33). ASR is one of the most destructive foliar diseases of soybean (Glycine max) and has been known to drastically reduce the yield by causing severe defoliation (14). The threat of widespread infections of soybean during the growing season (July to November) has increased in the past few years since the pathogen, P. pachyrhizi, has a wide host range and is capable of overwintering on a number of alternative hosts including kudzu (Pueraria lobata) leading to high inoculum accumulation (24,26). The disease is mostly restricted to the Southern states due to the mild winter conditions, which are favorable for the ASR establishment (26,30). In general, the environmental conditions, such as temperatures ranging between 15-26°C, humidity as high as 80%, and good growth and full canopy development of the soybean crop, are most suitable to promote the development of ASR (19). Currently, the control of ASR is mainly through fungicide applications. Cultural practices have also been recommended to reduce the impact of the pathogen on the crop (9). However, continuous increase in the use of fungicides 25 November 2013 Plant Health Progress has led to several serious problems, such as fungicide resistance and toxicity to non-target aquatic organisms in the streams or ponds near the fields sprayed with the fungicides (23). Fungicides also have shown toxic effect to mammalian cells based on a laboratory assay (7). For these above reasons, improving host resistance of soybeans to ASR is considered the most viable alternative approach to fungicide applications. As a long term control measure, efforts in screening for rust resistant soybean lines have identified six major R genes, Rpp1 to Rpp6 (13,20), that confer resistance to certain specific isolate(s) of P. pachyrhizi (1,27,29). This is due to high genetic variation among different populations of P. pachyrhizi (11,38). Therefore, developing durable genetic resistance against ASR has been difficult. Recently, molecular based approaches have been used to understand the mechanisms of host-pathogen interactions and to identify the genes involved in host defense responses to ASR. Several microarray analyses of host responses to ASR have been reported (5,8,25,34,37) and many up-regulated genes associated with basic defense and down-regulated genes associated with metabolic pathways in the rust-infected susceptible soybean palisade and mesophyll cells have been identified. However, little information can be inferred from the microarray studies as to how these rust-induced genes respond at the protein level. Therefore, a proteomics-based investigation of host defenses is necessary to have a better understanding of how soybean responds to rust infection at the