<i>Medicago truncatula</i> symbiosis mutants affected in the interaction with a biotrophic root pathogen (2014)

Understanding how plants balance between enabling microbial symbionts and fending off pathogens has direct implications both for basic plant biology and optimal use of crop plants in agriculture. The degree to which the processes associated with these two types of interactions overlap is poorly known. Recent studies revealed that symbiotic and pathogenic filamentous microbes require common plant genetic elements to establish colonization (Wang et al., 2012; Rey et al., 2013), supporting the long-held view that plants have evolved the ability to accommodate microbes (Parniske, 2000) and that pathogens have exploited these pathways. However, the extent to which plant genes implicated in fungal or bacterial symbioses are involved in interactions with biotrophic pathogens is unknown and research has been hampered by the lack of suitable common host experimental systems. Phytophthora palmivora, a root-infecting oomycete, is an aggressive biotrophic pathogen of Medicago truncatula, a model legume plant that is widely used in symbiosis research. Expression of fluorescent proteins in P. palmivora permits visualization of infection structures in M. truncatula roots. During its initial biotrophic colonization of M. truncatula roots which lasts until c. 48 h post-inoculation (hpi), P. palmivora zoospores expressing tdtomato red fluorescent protein (isolate AJ-td) germinate and form appressoria to penetrate the epidermis followed by rapid colonization of the root cortex apoplast and projection of short specialized hyphae, termed haustoria into plant cells (Fig. 1a). P. palmivora infection is accompanied by visible disease development in M. truncatula seedlings consisting of translucent tissue at the root tip at 2 d post-inoculation (dpi) and tissue browning in upper parts of the root at 3 dpi. (Fig. 1b, see also Supporting Information Methods S1). Concomitant with a switch to a nectrophic lifestyle, the pathogen enters the vasculature (Fig. 1a). In this study, we took advantage of a newly established quantitative Phytophthora palmivora–Medicago truncatula system to assess the extent to which mutants perturbed in colonization by arbuscular mycorrhizal fungi (AM fungi) and/or bacterial root nodule symbiosis are affected in the early/biotrophic stages of oomycete pathogenesis (Table S1). We devised and implemented a high throughput seedling infection assay and applied it to 19 M. truncatula lines mutated in 14 genes (for details see Methods S1; for explanation of gene abbreviations see Table S1). We measured both the overall root length and disease development, then plotted them as a ratio (Figs 1a,c, S1; Table S2). Of the 14 genes tested, seven (nine alleles) showed an altered response to P. palmivora inoculation compared with the wild-type Jemalong A17. Mutants in RAM2 and NIP/LATD showed enhanced resistance whereas mutants in five genes, NFP, LYK3, ERN, EFD, and LIN, all of which are impaired in the interaction with nitrogen fixing rhizobia displayed enhanced susceptibility. Expression levels of two defence response genes in M. truncatula mutants with altered disease symptoms were not overall significantly different from levels observed during infection of wild-type A17 seedlings (Fig. S2). This suggests that observed differences in disease extent are not attributable to altered defence responses in these mutants. These findings reveal a significant overlap between processes that define symbiosis and disease in M. truncatula roots. However, the remaining M. truncatula mutants unaltered in P. palmivora disease development include the common symbiotic signalling pathway (CSSP) mutants dmi1, dmi2 and dmi3, suggesting that the CSSP is not a major modulator of susceptibility to P. palmivora in M. truncatula. The mutants nfp-1, nfp-2 and hcl-2 encode defective NFP and LYK3 LysM domain receptor-like kinases (LysM-RLKs) (Table S1). NFP and LYK3 are required for plant responses to lipochitooligosaccharides (LCOs) from root nodule bacteria and mutants in both genes are impaired in nodule formation (Arrighi et al., 2006; Smit et al., 2007). Furthermore, nfp-1 is affected in transcriptional responses to fungal and bacterial LCOs (Czaja et al., 2012). Interestingly, nfp-1, nfp-2 and the LYK3 mutant hcl-2 showed enhanced disease symptoms during P. palmivora infection (Fig. 1c). Rey et al. (2013) previously reported that nfp mutants displayed enhanced susceptibility to the P. palmivora unrelated oomycete Aphanomyces euteiches and the fungal pathogen Colletotrichum trifolii. Our data further strengthens a role for NFP as negative regulator in plant interactions with a biotrophic filamentous pathogen. Given that both, NFP and LYK3 are involved in bacterial LCO perception, we tested whether lyk3/hcl mutants are also displaying enhanced susceptibility to P. palmivora. We found, that hcl-2 but not hcl-1 was altered in disease development. The hcl-1 allele harbours a missense mutation in the conserved kinase I motif presumably rendering it nonfunctional. Conversely, hcl-2 carries a splice site mutation giving rise to transcripts potentially encoding severely truncated LYK3 proteins or a full length protein with significant part of LysM 3 domain deleted (Smit et al., 2007). It is possible that truncated LYK3 proteins specifically interfere with defence signalling elements involved in P. palmivora perception. The U-Box/WD40 LIN protein functions during rhizobial colonization and abortion of this process in lin-2 leads to a suppression of nodule development (Kuppusamy et al., 2004; Kiss et al., 2009). Identification of LIN targets and their functional tests in P. palmivora interactions should aid future dissection of common and specific regulatory nodes for root nodule development and pathogen colonization. Taken together, our results thus highlight the need for understanding which are the common mechanisms involved in symbiosis and disease development. We studied transcription factors and their target genes to address whether the regulatory networks identified in symbiosis also apply for P. palmivora disease development. Expression of symbiosis genes in part is regulated by GRAS-type transcription factors such as RAM1 and NSP1 and NSP2 which act as heterodimers. RAM1/NSP2 (Gobbato et al., 2012) regulate RAM2 expression and NSP1/NSP2 activate ERN1 (Cerri et al., 2012). Mutations in RAM1, NSP1, NSP2 or ERNs are all impaired in either mycorrhiza or nitrogen fixing symbiosis (see references in Table S1). Here we confirmed requirement of RAM2 for P. palmivora infection. Moreover, we found that ERN1 negatively regulates P. palmivora disease development. However, mutations in the RAM2 and ERN1 expression regulating transcription factors RAM1 and NSP1 are dispensable for P. palmivora infection (Fig. 1c). This suggests that symbiosis and disease responses to P. palmivora may be integrated into common elements via different regulatory nodes resulting in opposite outcomes. Taken together, the disease phenotypes we report here should help the understanding of the biological processes perturbed in symbiosis mutants. Phytophthora, a genus of destructive plant pathogens of global importance to agriculture, is formed primarily of species that cause disease on plant roots. Yet only a few plant mutants affected in response to root infection by Phytophthora pathogens have been reported. Here, we implicate seven genes in disease development to P. palmivora in roots and gained new insight on the host genetic requirement for limiting pathogen infection. Two mutants showed enhanced resistance to P. palmivora and ram2-1 has previously been demonstrated to be more resistant to P. palmivora as well as A. euteiches (Wang et al., 2012; Gobbato et al., 2013). However, both ram2-1 and latd mutants still display a limited degree of colonization, likely attributable to the ability of Phytophthora species to utilize alternative infection routes which do not require appressorium mediated cell penetration such as penetration through middle lamellae of anticlinal host cell walls (Hardham, 2001) or through wounds (Drenth & Guest, 2004). We assessed whether ram2-1 and latd are impaired in haustorium formation using P. palmivora strains expressing fluorescent proteins. We detected presence of haustoria in both, latd and ram2-1 (Figs 1d, S4) as well as in all other tested mutants (Fig. S3). Notably, ram2-1 is strongly impaired in arbuscule formation (Wang et al., 2012). We speculate that both, latd and ram2-1 are impaired in entering the root resulting in a lower overall infection efficiency. RAM2 has been reported to be impaired in production of cutin monomers required for appressorium formation as well as seed coat processes (Wang et al., 2012). The mechanistic basis for resistance in latd remains to be studied but possibly points to a role of plant hormone and reactive oxygen species (ROS) regulated processes in the interaction between P. palmivora and M. truncatula. ROS levels are elevated in latd mutants (Zhang et al., 2014). The LATD gene encodes a predicted NRT1(PTR) transporter (Yendrek et al., 2010), and the latd mutant (W341STOP) has defects in root architecture including root hair defects and lateral roots do not fully differentiate. Mtlatd also has defective nodules (Bright et al., 2005). Mutant latd plants have wild-type levels of endogenous abscisic acid (ABA), but exhibit reduced sensitivity to the effect of ABA on stimulation of both stomatal closure and inhibition of seed germination (Liang et al., 2007). Future experiments should address whether high levels of ABA can complement the P. palmivora disease symptom phenotype in a similar manner to ABA treatment reversed ROS accumulation (Zhang et al., 2014). EFD is another modulator of hormone signalling, that was found to be important for P. palmivora infection. EFD is an ERF transcription factor which negatively regulates nodule development through interference with cytokinin signalling and mutants result in a higher number of nodules defective in nitrogen fixation (Vernié et al., 2008). Likewise, we observed extended disease symptom length with P. palmivora in efd-1 plants. Given, that efd-1 is not affected in colonization by AM fungi, we hypothesize that EFD modulates symptom development rather than early colonization by P. palmivora. Considering that EFD affects a cytokinin signalling response regulator, future work should assess the importance of cytokinin signalling on root colonization by P. palmivora. Taken together, our data implies a role for the plant hormones ABA and cytokinin in disease development. Our finding, that seven out of 14 genes implicated in symbiosis also impact P. palmivora disease development highlights that several plant symbiosis genes are involved in interactions with biotrophic pathogens. This is consistent with the long-held view that plants have evolved the ability to accommodate microbes and that pathogens have exploited these pathways (Parniske, 2000). However, new questions arise from this work. Five genes exert opposing roles in symbiosis vs disease development. Perhaps, plants utilize these genes to balance between defence and symbiosis. Surprisingly, mainly nitrogen fixation mutants but not mycorrhiza mutants were affected in P. palmivora infection. This might highlight an overlap in general cellular and developmental processes which have not been picked up by the slower and often less sensitive AM fungus colonization assays. Also, several mutants used in this study remain to be thoroughly characterized for their mycorrhizal phenotype. It is conceivable, that testing of all available symbiosis mutants with biotrophic pathogens will further our understanding on the overlap of molecular mechanisms underpinning symbiosis and disease development. Assessing their role in disease development in plant species which only support one type of symbiosis, mycorrhiza or nitrogen fixing symbiosis could enable further disentanglement of molecular pathways. Genetic and biochemical characterization of proteins with opposing outcomes will provide inroads to engineering disease resistant plants which retain symbiosis capabilities. The authors wish to thank numerous members of the symbiosis community for providing plant material and fruitful discussions (see Table S1), S. Whisson and H. Judelson for providing fluorescent protein expression vectors and M. Smoker and T. Yunusov for help with Phytophthora palmivora culturing and transformation. The authors are grateful to S. Kamoun for his continued support and input into an early draft of this manuscript. This work was supported by the Gatsby Charitable Foundation (RG62472 to S.S.); by the Royal Society (RG69135 to S.S.) and by a Marie Curie Intra-European fellowship (FP7-PEOPLE-2013-IEF, SYMUNITY-EC project 624398 to T.R.). Please note: Wiley Blackwell are not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office. Fig. S1 Average root length and average length of symptomatic tissues upon infection by Phytophthora palmivora 2 d post-inoculation. Fig. S2 Induction of defence associated genes in mutants and A17 wild-type (WT) upon infection by Phytophthora palmivora. Fig. S3 Medicago truncatula symbiosis mutants permit haustorium formation by Phytophthora palmivora. Fig. S4 Magnified single confocal plane images of haustoria. Table S1 Brief description of Medicago truncatula mutants used in this study Table S2 Data obtained from disease extent scoring Methods S1 Biological material, growth conditions, inoculation assays, microscopy and gene expression analysis. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.