Identification of Kernel Proteins Associated with the Resistance to <i>Fusarium</i> Head Blight in Winter Wheat (<i>Triticum aestivum</i> L.)

<div><p>Numerous potential components involved in the resistance to <i>Fusarium</i> head blight (FHB) in cereals have been indicated, however, our knowledge regarding this process is still limited and further work is required. Two winter wheat (<i>Triticum aestivum</i> L.) lines differing in their levels of resistance to FHB were analyzed to identify the most crucial proteins associated with resistance in this species. The presented work involved analysis of protein abundance in the kernel bulks of more resistant and more susceptible wheat lines using two-dimensional gel electrophoresis and mass spectrometry identification of proteins, which were differentially accumulated between the analyzed lines, after inoculation with <i>F. culmorum</i> under field conditions. All the obtained two-dimensional patterns were demonstrated to be well-resolved protein maps of kernel proteomes. Although, 11 proteins were shown to have significantly different abundance between these two groups of plants, only two are likely to be crucial and have a potential role in resistance to FHB. Monomeric alpha-amylase and dimeric alpha-amylase inhibitors, both highly accumulated in the more resistant line, after inoculation and in the control conditions. <i>Fusarium</i> pathogens can use hydrolytic enzymes, including amylases to colonize kernels and acquire nitrogen and carbon from the endosperm and we suggest that the inhibition of pathogen amylase activity could be one of the most crucial mechanisms to prevent infection progress in the analyzed wheat line with a higher resistance. Alpha-amylase activity assays confirmed this suggestion as it revealed the highest level of enzyme activity, after <i>F. culmorum</i> infection, in the line more susceptible to FHB.</p></div

Comparison of alpha-amylase activity in the kernels of winter wheat (<i>Triticum aestivum</i>) SL (line more susceptible to <i>Fusarium</i> head blight) and RL (line more resistant to <i>Fusarium</i> head blight) after <i>Fusarium culmorum</i> infection (<i>Fusarium</i>-damaged kernels) and in control conditions.

<p>The enzyme activity was expressed in Ceralpha Units (CU) per gram of flour. The standard deviation bars are shown.</p

Comparison of selected kernel protein abundance after <i>Fusarium culmorum</i> infection and in the control conditions in the winter wheat (<i>Triticum aestivum</i>) SL (line more susceptible to <i>Fusarium</i> head blight) and the RL (line more resistant to <i>Fusarium</i> head blight).

<p>Spot numbering is the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110822#pone-0110822-g002" target="_blank">Fig. 2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110822#pone-0110822-g003" target="_blank">3</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110822#pone.0110822.s001" target="_blank">S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110822#pone.0110822.s002" target="_blank">S2</a>. The standard deviation bars are shown. Only proteins identified from homogenous spots are shown.</p

Diagram demonstrating a workflow of sample preparation for proteome analysis.

<p>Abbreviations: FDK, <i>Fusarium</i>-damaged kernels; HLK, healthy-looking kernels; RL, line of winter wheat more resistant to <i>Fusarium</i> head blight; SL, line of winter wheat more susceptible to <i>Fusarium</i> head blight.</p

One representative 2-DE protein map of winter wheat (<i>Triticum aestivum</i>) kernel after <i>Fusarium culmorum</i> infection (<i>Fusarium</i>-damaged kernels) for the line more susceptible (SL) to <i>Fusarium</i> head blight.

<p>The spots with differentially accumulated (p≤0.05) proteins (1–9) identified in the SL, are circled with a solid line. Molecular weight (MW) scale is shown.</p

The components of the resistance to <i>Fusarium</i> head blight in the more resistant (RL) and more susceptible (SL) winter wheat (<i>Triticum aestivum</i>) lines and their yields under control conditions.

<p>FHBi – <i>Fusarium</i> head blight index, FDK – <i>Fusarium</i>-damaged kernels, RL – more resistant line, SL – more susceptible line; mean values and standard deviations of each parameter calculated after inoculation (three plots) and data from one plot calculated for the control conditions, are shown.</p><p>The components of the resistance to <i>Fusarium</i> head blight in the more resistant (RL) and more susceptible (SL) winter wheat (<i>Triticum aestivum</i>) lines and their yields under control conditions.</p

Image_2_Insight into metabolic sensors of nitrosative stress protection in Phytophthora infestans.jpeg

Phytophthora infestans, a representative of phytopathogenic oomycetes, have been proven to cope with redundant sources of internal and host-derived reactive nitrogen species (RNS). To gain insight into its nitrosative stress resistance mechanisms, metabolic sensors activated in response to nitrosative challenge during both in vitro growth and colonization of the host plant were investigated. The conducted analyses of gene expression, protein accumulation, and enzyme activity reveal for the first time that P. infestans (avirulent MP946 and virulent MP977 toward potato cv. Sarpo Mira) withstands nitrosative challenge and has an efficient system of RNS elimination. The obtained data indicate that the system protecting P. infestans against nitric oxide (NO) involved the expression of the nitric oxide dioxygenase (Pi-NOD1) gene belonging to the globin family. The maintenance of RNS homeostasis was also supported by an elevated S-nitrosoglutathione reductase activity and upregulation of peroxiredoxin 2 at the transcript and protein levels; however, the virulence pattern determined the expression abundance. Based on the experiments, it can be concluded that P. infestans possesses a multifarious system of metabolic sensors controlling RNS balance via detoxification, allowing the oomycete to exist in different micro-environments flexibly.</p

Image_5_Insight into metabolic sensors of nitrosative stress protection in Phytophthora infestans.tif

Phytophthora infestans, a representative of phytopathogenic oomycetes, have been proven to cope with redundant sources of internal and host-derived reactive nitrogen species (RNS). To gain insight into its nitrosative stress resistance mechanisms, metabolic sensors activated in response to nitrosative challenge during both in vitro growth and colonization of the host plant were investigated. The conducted analyses of gene expression, protein accumulation, and enzyme activity reveal for the first time that P. infestans (avirulent MP946 and virulent MP977 toward potato cv. Sarpo Mira) withstands nitrosative challenge and has an efficient system of RNS elimination. The obtained data indicate that the system protecting P. infestans against nitric oxide (NO) involved the expression of the nitric oxide dioxygenase (Pi-NOD1) gene belonging to the globin family. The maintenance of RNS homeostasis was also supported by an elevated S-nitrosoglutathione reductase activity and upregulation of peroxiredoxin 2 at the transcript and protein levels; however, the virulence pattern determined the expression abundance. Based on the experiments, it can be concluded that P. infestans possesses a multifarious system of metabolic sensors controlling RNS balance via detoxification, allowing the oomycete to exist in different micro-environments flexibly.</p

Image_1_Insight into metabolic sensors of nitrosative stress protection in Phytophthora infestans.tif

Phytophthora infestans, a representative of phytopathogenic oomycetes, have been proven to cope with redundant sources of internal and host-derived reactive nitrogen species (RNS). To gain insight into its nitrosative stress resistance mechanisms, metabolic sensors activated in response to nitrosative challenge during both in vitro growth and colonization of the host plant were investigated. The conducted analyses of gene expression, protein accumulation, and enzyme activity reveal for the first time that P. infestans (avirulent MP946 and virulent MP977 toward potato cv. Sarpo Mira) withstands nitrosative challenge and has an efficient system of RNS elimination. The obtained data indicate that the system protecting P. infestans against nitric oxide (NO) involved the expression of the nitric oxide dioxygenase (Pi-NOD1) gene belonging to the globin family. The maintenance of RNS homeostasis was also supported by an elevated S-nitrosoglutathione reductase activity and upregulation of peroxiredoxin 2 at the transcript and protein levels; however, the virulence pattern determined the expression abundance. Based on the experiments, it can be concluded that P. infestans possesses a multifarious system of metabolic sensors controlling RNS balance via detoxification, allowing the oomycete to exist in different micro-environments flexibly.</p

Table_1_Insight into metabolic sensors of nitrosative stress protection in Phytophthora infestans.docx

Phytophthora infestans, a representative of phytopathogenic oomycetes, have been proven to cope with redundant sources of internal and host-derived reactive nitrogen species (RNS). To gain insight into its nitrosative stress resistance mechanisms, metabolic sensors activated in response to nitrosative challenge during both in vitro growth and colonization of the host plant were investigated. The conducted analyses of gene expression, protein accumulation, and enzyme activity reveal for the first time that P. infestans (avirulent MP946 and virulent MP977 toward potato cv. Sarpo Mira) withstands nitrosative challenge and has an efficient system of RNS elimination. The obtained data indicate that the system protecting P. infestans against nitric oxide (NO) involved the expression of the nitric oxide dioxygenase (Pi-NOD1) gene belonging to the globin family. The maintenance of RNS homeostasis was also supported by an elevated S-nitrosoglutathione reductase activity and upregulation of peroxiredoxin 2 at the transcript and protein levels; however, the virulence pattern determined the expression abundance. Based on the experiments, it can be concluded that P. infestans possesses a multifarious system of metabolic sensors controlling RNS balance via detoxification, allowing the oomycete to exist in different micro-environments flexibly.</p