Molecular cues of Cuscuta and their effects in host plants

Abstract

Parasitic flowering plants live as heterotrophs and obtain inorganic and organic nutrients from their hosts. C. reflexa, belonging to the genus Cuscuta comprising of approximately 200 species, is a holoparasite with the appearance of a thread-like vine that coils around its hosts shoot. During the infection process, C. reflexa forms a specialized haustorium for penetrating the hosts shoot and connecting to the hosts xylem and phloem. While nearly all plants are susceptible hosts for Cuscuta spp., tomato as one of few exceptions shows an active resistance against C. reflexa. The aims of this work were the identification of new resistance related genes in the resistant host S. lycopersicum and the identification of changes in phytohormone metabolism during the infection of C. reflexa on a susceptible host. A library of introgression lines between resistant S. lycopersicum and susceptible S. pennellii carrying different chromosomal recombinations, was screened for susceptibility and a hypersensitive response occurring at the attempted haustorium penetration sites. A region of about 172 kbps on chromosome 12 revealed a function in defence against C. reflexa. S. lycopersicum plants lacking this region showed susceptibility against the plant parasite. By excising smaller chromosomal parts from the region of interest with a CRISPR-Cas9 technique the number of potential candidate genes could be further narrowed down. The susceptibility mapped to chromosome 12 does not influence immune responses like ROS and ethylene production which are linked to the first identified resistance trait CuRe1. Single knock outs of the identified candidate genes will reveal the exact gene/genes responsible for the second resistance trait in S. lycopersicum against C. reflexa. A complementation with respected gene(s) will prove this. During the interaction of C. reflexa and a susceptible host, numerous signals may be exchanged to ensure a successful invasion through the parasite. Most likely, in this process there is a directed influence on phytohormone metabolism. This is to be expected with a high probability since these signalling molecules are vital for almost all processes inside the plant. To identify those changes several experimental approaches were tested, e.g. GCMS, LC-MS, fluorescence plant marker lines (COLORFUL lines) and hormonedeficient mutants. These techniques were all used to monitor phytohormone changes during an infection of susceptible A. thaliana or N. tabacum by C. reflexa over a timeof up to 10 days. Changes have been analysed for the phytohormones auxin, cytokinins, jasmonate, jasmonate/ethylene, abscisic acid and salicylic acid. None of those hormones showed consistent reproducible trends for up- or downregulation of metabolism. This may be due to several reasons, like the low total abundance of phytohormones in general and therefore the challenges of measuring their total content or due to an influence of the parasite on a different regulatory level (even different hormonal level). Even an active suppression of unfavourable hormones through the parasite during the infection is possible. A repetition of the experiments with a much higher number of plants could give better insights into slight changes of phytohormone levels. Also promising could be the analysis of other hormones not included in our studies, like e.g. brassinolide or peptide hormones or even a comparison on genetic level with established marker genes.