Untersuchungen zum Infektionsmechanismus des Asiatischen Sojabohnenrostpilzes Phakopsora pachyrhizi auf seiner Wirtpflanze Sojabohne
- Studies on the infection mechanism of the Asian soybean rust fungus Phakopsora pachyrhizi on its host plant soybean
Löhrer, Marco; Schaffrath, Ulrich (Thesis advisor); Panstruga, Ralph (Thesis advisor)
Dissertation / PhD Thesis
Dissertation, RWTH Aachen University, 2016
<i>Phakopsora pachyrhizi</i> SYD. & P. SYD, the causal agent of Asian soybean rust is one of the economically most important pathogens on soybean. Plants resistant against all isolates of the fungus are not yet available and it is foreseeable that resistances against fungicides in use will occur. Therefore, this study aimed at gaining a deeper understanding of the infection mechanism with the goal of identifying novel and innovative strategies to combat the disease. Methods applied to reach that goal ranged from histology and microscopy to molecular biology.The first critical stage during the invasion of host plants by <i>P. pachyrhizi</i> is the penetration of the host tissue. In this study, it could be shown that the unusual direct penetration of <i>P. pachyrhizi</i> urediospores is mediated via a high turgor pressure in the hyaline, non-melanized appressoria. To achieve this, Mach-Zehnder transmission- light interferometry was applied. On basis of the "mean specific optical path difference" the concentration of substances in the cytosol of <i>P. pachyrhizi</i> and <i>M. oryzae</i> appressoria were compared. GC-TOF/MS measurements allowed the identification of appressorial cytosolic compounds, which were needed to determine the refractive index increments. Using this parameter, the osmotic potential of the cytosolic content of <i>P. pachyrhizi</i> and <i>M. oryzae</i> appressoria could be calculated to 3.9 MPa and 6.75 MPa, respectively. Incipient cytorrhysis was used as a method for independent verification of these results. Experiments on inert PTFE membranes showed that the pressure in the soybean rust appressoria alone is sufficient for penetration of membranes of thicknesses in the range of the soybean leaf cuticle (130 nm).Studies on the genome and transcriptome of <i>P. pachyrhizi</i> were conducted to answer the question which additional factors enable the penetration of the host plant and the establishment of the biotrophic interaction. Partial sequencing followed by K-mer analysis allowed the estimation of the size of the soybean rust genome to around 1 Gb and suggested a high degree of heterozygosity. Based on these results an Illumina-RNA-seq approach was followed. Early infection stages including appressoria and isolated fungal haustoria from late infection stages were sampled and RNA was extracted and sequenced. The <i>de novo</i> assembled transcriptome was comprised of 29.362 sequences with a N50-value of 2868 bp which could be narrowed down to 20.612 sequences via in silico translation and redundancy filtering. The differential expression analysis between appressorial- and haustorial stages revealed distinct groups of effectors and cell wall degrading enzymes. The de novo assembled transcriptome was further used for nano-LC-MS/MS based identification of proteins in the urediospore germination fluids. Among these proteins, those carrying a signal peptide were significantly enriched and cell wall degrading enzymes and effectors could be identified.Since stable deletion or silencing of genes is not yet possible in the obligate biotrophic fungus <i>P. pachyrhizi</i>, alternative methods for functional gene characterization were evaluated. Down-regulation of mRNA abundance via exogenous applied small interfering RNA (siRNA) or siRNA formed <i>in planta</i> were tested. Remarkably, it was possible to decrease germ tube length by transcriptional silencing of a cell wall biosynthesis associated <i>beta-1,3-glucan-synthase</i> gene Agrobacterium-mediated expression of a <i>beta-1,3-glucan-synthase</i>- and a <i>sterol-14-alpha-demethylase</i>-hairpin construct in planta led to decreased fungal growth after inoculation. The novel insights into the infection mechanism obtained in this study and the <i>de novo</i> sequenced and assembled transcriptome are a basis for further studies aiming at minimizing <i>P. pachyrhizi’s</i> detrimental impact on soybean production.