Proteome Changes in Stem Tissues of Sunflower Lines Inoculated with Culture Filtrate of Sclerotinia sclerotiorum

Abstract

Background: Sclerotinia sclerotiorum (Lib.) de Bary cause a deleterious disease on sunflower plants. Oxalic acid is the main pathogenicity factor of S. sclerotiorum. Two dimensional gel electrophoresis and mass spectrometry have been used in several studies to investigate molecular changes that occur in the plants in response to S. sclerotiorum infection. Comparing responses of resistant and susceptible lines upon pathogen infection provided novel information regarding defense mechanisms against this necrotrophic pathogen.

Objectives: The present study reports proteome changes of partially resistant and susceptible sunflower lines under pathogen's culture filtrate treatment, resulting in the characterization of up- and down- regulated proteins.

Material and methods: Sunflower partially resistant and susceptible lines with two true leaves were exposed to fungus culture filtrate. The stems of treated and untreated plants were sampled at 24, 48 and 72 hours after treatment for two-dimensional electrophoresis. Twenty spots showed more than 1.5-fold change in abundance were subjected to MALDI/TOF-TOF MS for further analysis.

Results: The identified proteins were categorized into several classes including carbohydrate and energy metabolism (25%), cellular metabolic process (15%), stress response (15%), plant cell wall biogenesis (10%), photosynthesis (10%), protein metabolism (10%), unknown function (10%) and redox homeostasis (5%).

Conclusions: Our proteomic investigation demonstrates an increase in the expression of proteins only in partially resistant line, such as proteins involved in carbohydrate metabolism and plant defense responses (malate dehydrogenase and peroxidase), metabolic process (adenosine kinase), regulating cell redox homeostasis (disulfide isomerase) and lignin biosynthetic process (laccase). Moreover, the expression of pyrroline-5-carboxylate reductase, involved in proline biosynthesis, was significantly changed in both sunflower lines in response to pathogen culture filtrate. Proteins which were only up-regulated in the partially resistant lines might have a significant role in mediating the defense against Sclerotinia and could be considered for enhancing resistance against this devastating pathogen.

Keywords: 2-DE; Biotic stress; Sunflower; Tolerance; Sclerotinia.

Figure 1

Representative image of partially resistant or susceptible line stem proteins separated by 2-DE. The 2-DE was performed for both lines at 24, 48 and 72 h post treatment by using 7 cm IPG strips and 12% SDS-PAGE gels. Gels were stained with Coomassie Brilliant Blue R-250. Numbers and arrows shows significantly altered spots and their descriptions have been shown in Table 1.

Figure 2

Closer view of protein spots in susceptible (S) and partially resistant (R) lines at different time points (24, 48 and 72 h post treatment) compared to non-inoculated samples (c). Panel (I) indicates those spots that changed at one time point. Panel (II) and (III) indicate those spots that changed at more than one time point

Figure 3

Main metabolic pathways changed in sunflower stem in response to virulence factors of S. sclerotiorum. The color graphs indicate fold change of identified proteins at various time points. Columns from left to right represent results obtained from various sampling times (24, 48 and 72 h after treatment). The first row represents results for the susceptible line and the second row represents results for the partially resistant line. Color scales of fold change values are shown, in which red and green colors indicate the higher and lower expression levels, respectively. Column chart shows SKDH activity in partially resistant (R) and susceptible (S) lines after treatment by pathogen culture filtrate ( 25 ). Line graph shows relative activity of PAL and column chart links to proline content in S and R lines at various time points ( 26 ). R, partially resistant line AC4122; S, susceptible line HA89; DHAP, dihydroxy acetone phosphate; HSP70, heat shock 70 kDa protein; Malate DH, malate dehydrogenase; PAL, phenylalanine ammonia lyase; PDI, protein disulfide-isomerase; SKDH, shikimate dehydrogenase; VDAC; voltage-dependent anion channel; NS, non-significant.

Figure 4

The functional classification of the identified proteins