Analysis of Phakopsora pachyrhizi transcript abundance in critical pathways at four time-points during infection of a susceptible soybean cultivar using deep sequencing

Abstract

Background: Phakopsora pachyrhizi, the causal agent responsible for soybean rust, is among the top hundred most virulent plant pathogens and can cause soybean yield losses of up to 80% when appropriate conditions are met. We used mRNA-Seq by Illumina to analyze pathogen transcript abundance at 15 seconds (s), 7 hours (h), 48 h, and 10 days (d) after inoculation (ai) of susceptible soybean leaves with P. pachyrhizi to gain new insights into transcript abundance in soybean and the pathogen at specific time-points during the infection including the uredinial stage.

Results: Over three million five hundred thousand sequences were obtained for each time-point. Energy, nucleotide metabolism, and protein synthesis are major priorities for the fungus during infection and development as indicated by our transcript abundance studies. At all time-points, energy production is a necessity for P. pachyrhizi, as indicated by expression of many transcripts encoding enzymes involved in oxidative phosphorylation and carbohydrate metabolism (glycolysis, glyoxylate and dicarboxylate, pentose phosphate, pyruvate). However, at 15 sai, transcripts encoding enzymes involved in ATP production were highly abundant in order to provide enough energy for the spore to germinate, as observed by the expression of many transcripts encoding proteins involved in electron transport. At this early time-point, transcripts encoding proteins involved in RNA synthesis were also highly abundant, more so than transcripts encoding genes involved in DNA and protein synthesis. At 7 hai, shortly after germination during tube elongation and penetration, transcripts encoding enzymes involved in deoxyribonucleotide and DNA synthesis were highly abundant. At 48 hai, transcripts encoding enzymes involved in amino acid metabolism were highly abundant to provide for increased protein synthesis during haustoria maturation. During sporulation at 10 dai, the fungus still required carbohydrate metabolism, but there also was increased expression of transcripts encoding enzymes involved in fatty acid metabolism.

Conclusion: This information provides insight into molecular events and their timing throughout the life cycle of the P. pachyrhizi, and it may be useful in the development of new methods of broadening resistance of soybean to soybean rust.

Figure 1

Representation of the glycolysis pathway in which regulation status of transcripts encoding enzymes identified at (A) 7 hai, compared to 15 sai, (B) 48 hai, compared to 7 hai, and (C) 10 dai, compared to 48 hai, is annotated. Boxes colored in red represent enzymes encoded by down-regulated transcripts; boxes colored in green represent enzymes encoded by up-regulated transcripts; while boxes colored in yellow represent enzymes encoded by transcripts with varied regulatory status. Boxes colored in black represent enzymes encoded by transcripts identified at the experimental time-point but not at the previous time point.

Figure 2

Representation of the oxidative phosphorylation pathway in which regulation status of transcripts encoding enzymes identified at (A) 7 hai, compared to 15 sai, (B) 48 hai, compared to 7 hai, and (C) 10 dai, compared to 48 hai, is annotated. Color coding as in Figure 1.

Figure 3

Representation of arginine and proline metabolism in which regulation status of transcripts encoding enzymes identified at (A) 7 hai, compared to 15 sai, (B) 48 hai, compared to 7 hai, and (C) 10 dai, compared to 48 hai, is annotated. Color coding as in Figure 1.

Figure 4

Functional categorization of potential new Phakopsora pachyrhizi transcripts identified at (A) 15 sai, (B) 7 hai, (C) 48 hai, and (D) 10 dai following protein and conserved domain similarity searches against various databases. The percentage of potential new transcripts found in each category is represented.

Figure 5

Schematic representation of events occurring in the pathogen during the infection process; A) 15 sai; B) 7 hai, C) 48 hai, and D) 10 dai of a susceptible soybean leaf with Phakopsora pachyrhizi. Drawing in black represents a cross-section of a soybean leaf where zone 1 represents the upper cuticle, zone 2 the upper epidermis cell layer, zone 3 the palisade mesophyll cell layer, zone 4 the spongy mesophyll cell layer, zone 5 the lower epidermis cell layer, and zone 6 the lower cuticle. Drawing in brown represents P. pachyrhizi structures on and inside the soybean leaf showing spore (S), mitochondrion (M), germ tube (GT), appressorium (A), primary hypha (PH), infectious hyphae (IH), haustorium (H), and uredinium (U). Metabolic pathways, proteins, E. C. numbers, products, and substrates colored in blue are activated or expressed; those colored in green are up-regulated compared to the previous time-point; and those colored in red are down-regulated compared to the previous time-point; while boxes colored in yellow represent enzymes encoded by transcripts with varied regulatory status. Metabolic pathways, proteins, E.C. numbers, products, and substrates colored in black were not activated or expressed in the present data set.