Using genome diversity to decipher nematode virulence and host susceptibility

The sedentary root-knot nematode Meloidogyne incognita is a widely distributed and highly polyphagous phytopathogen, which causes annual losses in the order of millions of dollars in damage to crops. M. incognita juveniles initiate the development of a permanent feeding site consisting of so-called giant cells. These giant cells nourish the nematodes during their life, while cells surrounding the giant cells are also hypertrophic and hyperplastic and form a large protective gall. The elaborate changes in plant roots leading to the formation of feeding sites are orchestrated by effectors in secretions of M. incognita. To introduce the current concepts of this close interaction at a molecular level in Chapter 1, the latest progress with regard to identification and functional characterization of M. incognita effectors is summarized. Furthermore, it is explained how effectors can play a role in the adaptive evolution of nematodes and hosts.

Chapter 2 describes the identification of the effector MiMSP32 based on specific patterns of genetic variation in the M. incognita genome. As a consequence of adaptive evolution, an ancestral gene of MiMSP32 gene has duplicated and diversified into a gene family with at least thirty identified variants, all taxonomically restricted to root-knot nematodes. These gene variants can be subdivided into six clusters based on their similarities. As a pioneer gene, MiMSP32 shows no similarity to any other functionally characterized genes or proteins However, we used the predicted secondary structure to identify a remote homology with several proteins adopting three-layer beta-alpha-beta (βαβ)-sandwich architecture. Based on the positive selection and gene expansion, we hypothesize that MiMSP32 has undergone functional diversification.

In Chapter 3, we study the biological relevance of MiMSP32 for infectivity of M. incognita on tomato plants. We functionally characterized MiMSP32 in planta and show that it is indeed an important effector with a role in nematode virulence and host plant susceptibility. Moreover, MiMSP32 proved to be a promiscuous effector, as we identified six host targets by screening a tomato cDNA library in yeast. We confirmed these interactions by multiple protein-protein interaction assays, such as co-immunoprecipitation, co-localization, and FRET-FLIM after transient expression in Nicotiana benthamiana leaves. From these results, a model emerges wherein the effector MiMSP32 promotes the virulence of M. incognita by interacting with multiple unrelated host proteins in tomato.

Next, we tested the susceptibility of existing T-DNA knock-out mutants of homologs of the six MiMSP32 host targets in Arabidopsis thaliana, which is a host of M. incognita. We show in Chapter 4 that the Arabidopsis knock-out opr2-1 mutant is significantly more susceptible to M. incognita than wild-type plants. AtOPR2 is thought to take part in an alternative jasmonic acid (JA) biosynthesis pathway downstream of 12-oxo-phytodienoate (OPDA) in the conversion of 4,5-didehydrojasmonate (4,5-ddh-JA) to JA, thereby suggesting that AtOPR2 may function in JA-dependent plant defense. However, our bioassays and transcriptional data provide evidence that AtOPR2 regulates susceptibility of Arabidopsis to M. incognita independent from basal plant immune responses by conversion of the signaling molecule 4,5-ddh-JA.

In Chapter 5, we describe an alternative approach to identify sources of tomato resistance to M. incognita. To this purpose, we used a collection of 178 domesticated tomato lines without known major R-genes to gauge the quantitative variation in tomato susceptibility to M. incognita. Next, we linked this trait to genomic regions of 156 of these tomato lines using a genome-wide association study (GWAS), resulting in a catalogue of 380 genes associated with tomato susceptibility to M. incognita. By using additional RNA-Seq of isolated nematode-induced galls on a representative subset of ten tomato accessions, we identified 37 differential regulated genes within the 380 gene candidates from the GWAS. These susceptibility-associated genes likely contain new sources of resistance for use in future studies and breeding applications.

In the final chapter of this thesis (Chapter 6), it is argued that genome diversity can help to identify key factors involved in the diversity of nematode virulence and host susceptibility. MiMSP32 was selected for further analyses based on positive, diversifying selection in the M. incognita genome. Likewise, the variation in the S. lycopersicum genome was used to identify genes significantly associated with quantitative variation in plant susceptibility. Host targets of positively selected nematode effectors are likely to generate a detectable genetic signal in studies of host susceptibility. To test this hypothesis, the 380 tomato susceptibility-associated genes (GWA) were compared with the 51 putative host target genes of MiMSP32 (Y2H). With this comparison, the hypothesis could not yet be confirmed, as the overlapping susceptibility-associated gene needs additional confirmation as a host target. However, confirmation of the hypothesis was possible based on the host target AtOPR2, as it regulates susceptibility of Arabidopsis to M. incognita. Therefore, a suggestion for future studies would be to integrate genome diversity of both nematode and host and use the obtained information of this thesis to formulate more efficient plant protection strategies