Transcriptional Adaptation of Mycosphaerella graminicola to Programmed Cell Death (PCD) of Its Susceptible Wheat Host

Abstract

Many important fungal pathogens of plants spend long periods (days to weeks) of their infection cycle in symptomless association with living host tissue, followed by a sudden transition to necrotrophic feeding as host tissue death occurs. Little is known about either the host responses associated with this sudden transition or the specific adaptations made by the pathogen to invoke or tolerate it. We are studying a major host-specific fungal pathogen of cultivated wheat, Septoria tritici (teleomorph Mycosphaerella graminicola). Here, we describe the host responses of wheat leaves infected with M. graminicola during the development of disease symptoms and use microarray transcription profiling to identify adaptive responses of the fungus to its changing environment. We show that symptom development on a susceptible host genotype has features reminiscent of the hypersensitive response, a rapid and strictly localized form of host programmed cell death (PCD) more commonly associated with disease-resistance mechanisms. The initiation and advancement of this host response is associated with a loss of cell-membrane integrity and dramatic increases in apoplastic metabolites and the rate of fungal growth. Microarray analysis of the fungal genes differentially expressed before and after the onset of host PCD supports a transition to more rapid growth. Specific physiological adaptation of the fungus is also revealed with respect to membrane transport, chemical and oxidative stress mechanisms, and metabolism. Our data support the hypothesis that host plant PCD plays an important role in susceptibility towards fungal pathogens with necrotrophic lifestyles.

Analysis of Two in Planta Expressed LysM Effector Homologs from the FungusMycosphaerella graminicolaReveals Novel Functional Properties and Varying Contributions to Virulence on Wheat

“…M. graminicola also penetrates wheat leaves through stomata but then only slowly colonizes the intercellular spaces of wheat leaves. However, this fungus appears to benefit from host cell death, which may provide sufficient nutrients to support its asexual sporulation (Kema et al, 1996;Keon et al, 2007;Deller et al, 2011). Early symptomless growth is slow, whereas growth rate during host cell death increases very rapidly, suggesting that M. graminicola is subject to initial nutrient limitation that is relieved once host cell permeability changes and the necrotrophic phase begins.…”

“…To determine the relative transcript levels for each MgLysM gene, real-time reverse transcription (RT)-PCR was performed on two in vitro growth samples and across five time points of wheat leaf infection, as follows: day 1, where fungal spores are germinating on the leaf surface; days 4 and 9, which span and extend to the limit of the symptomless intercellular growth phase; day 14, which corresponds to leaf cells undergoing cell death and releasing nutrients into the apoplast; and day 21, which corresponds to the fungus sporulating in fully necrotic leaf lesions (Keon et al, 2007). None of the MgLysM genes exhibited significant differential expression between the two in vitro growth conditions on Czapek-Dox broth (CDB) and PDB (Fig.…”

“…2C). As CDB facilitates slower fungal growth due to a poor nitrogen source (nitrate only; Keon et al, 2007), this suggests that MgLysM gene expression was not responsive to nitrogen limitation. No increase in expression for any MgLysM gene was detected at day 1 of plant infection.…”

“…For estimation of gene expression in vitro, the wild-type fungus was inoculated into cultures of PDB and CDB and shake flask cultures were harvested during logarithmic growth. Wheat (Triticum aestivum) leaf infection assays were performed as described previously (Keon et al, 2007).…”

“…Approximately 8 to 10 d after inoculation, disease lesions begin to form on susceptible plants (Kema et al, 1996). This transition is associated with the induction of host defense processes characteristic of hypersensitive response-like programmed cell death and differential regulation of wheat mitogen-activated protein kinase pathways (Keon et al, 2007;Rudd et al, 2008). These events culminate in loss of control of cell permeability, resulting in leakage of nutrients from dying plant cells into the apoplastic spaces where the fungus resides, and coincide with an exponential increase in its growth rate.…”