Analysis of the role of polygalacturonase inhibiting proteins in the interaction between plants and nematodes

Abstract

Plant-parasitic nematodes (PPNs) are a great threat to crops, causing billions of dollars of losses worldwide. Cyst nematodes and root-knot nematodes belong to a small group of sedentary endoparasitic PPNs that parasitize the roots of a wide range of crop species. Both of these nematodes invade the roots as infecting juveniles (J2s) and establish a<br /> feeding site in the plant root that functions as their nutrient source. An effective plant defence relies on the recognition of pathogen- or microbial-associated molecular patterns (PAMPs/MAMPs) by surface-localised pattern-recognition receptors (PRRs). In addition to PAMPs, PRRs can also recognize damage associated molecular patterns (DAMPs), which are molecules produced by plants in response to damage. Nematodes’ invasion of plant roots and their subsequent migration inside the roots are likely to damage the plant’s root cells, thereby generating cell wall fragments (oligogalacturonides OGs). OGs can act as DAMPs. Recognition of PAMPs or DAMPs by PRRs leads to the activation of defence responses in the host plant, which are designated as PAMP-triggered immunity (PTI). PTI can restrict development and growth of invading pathogens. Though nematode invasion activates PTI in plants during early stages of infection, PTI responses may in turn be suppressed by the invading nematodes during the establishment of their feeding sites. Recent studies have shown that plants use surface-localised receptors to recognise PAMPs released by nematodes, and thereby trigger PTI responses. However, recognition of DAMPs and activation of downstream pathways during plant-nematode interactions remained unexplored. In this study, we characterised the role polygalacturonase-inhibiting proteins (PGIP) in the model plant <em>Arabidopsis thaliana</em> during infection with the beet cyst nematode<em> Heterodera schachtii</em> and the root-knot nematode <em>Meloidogyne incognita</em>. PGIP is encoded in <em>Arabidopsis</em> by a small gene family consisting of two genes, <em>PGIP1</em> and <em>PGIP2</em>. Expression of these genes is induced when the plant is wounded or attacked by pathogens. In order to inhibit pectin degradation by microbial polygalacturonase (PG), the plant deploys PGIP in the cell wall. This PG-PGIP interaction leads to the production of elicitor-active OGs (oligomers of α-1, 4-linked galacturonic acids), which can be sensed by a plasma membrane localized receptor called <em>WAK1</em> (wall-associated kinase). The <em>WAK1</em> receptor, on sensing elicitor-activated OGs, sets off a number of defence responses in the plant such as accumulation of phytoalexins, oxidative burst, callose deposition, and production of glucanase and chitinase. Quantitative RT-PCR (qRT-PCR) analysis showed that <em>PGIP</em> genes are particularly strongly induced upon cyst nematode invasion of roots. To analyse spatio-temporal expression of <em>PGIP</em> genes during plant-nematode interaction, we developed promoter::GUS lines and observed strong staining at an early stage (1dpi) of <em>H. schachtii </em>infection. However, this staining was not observed when the plants were infected with <em>M. incognita</em> at the same stage. Pathogenicity testing with loss-of-function mutants (<em>pgip1, pgip2</em>) and overexpression lines (<em>35S::PGIP1, 35S::PGIP2</em>) revealed that <em>PGIP1</em> expression limits the ability of cyst but not root-knot nematodes to parasitize the host roots. A recent transcriptome analysis of host roots during the migratory stages of <em>H. schachtii</em> infection revealed that, during this stage, a number of genes involved in secondary metabolism (camalexin and indole glucosinolates production) were strongly and differentially upregulated. Because loss-of-function <em>PGIP1</em> mutants (<em>pgip1</em>) were hypersusceptible to beet cyst nematode infection, we reasoned that activation of the secondary metabolism genes in these mutants might be impaired. To investigate this hypothesis further, we compared Col-0 and <em>pgip</em> (<em>pgip1</em> and <em>pgip2</em>) roots for the expression of the secondary metabolism genes with or without infection via qRT-PCR. We found that induction of the secondary metabolism genes is impaired in pgip mutants, especially in <em>pgip1</em> during migratory stage. A detailed characterization of a putative PG sequence from <em>H. schachtii</em> showed a very low sequence similarity to a PG from <em>M. incognita</em> and any other known PG from bacteria, fungi, insects, and nematodes. Therefore, we concluded that cyst nematodes do not encode a functional PG and activation of PGIP during cyst nematode infection is independent of a typical PG-PGIP interaction. In conclusion, our findings provide insights into distinct perception of damage responses by host during cyst and root-knot nematode parasitism at the molecular level. Clarifying further details of these responses may lead to advances in breeding strategies for nematode resistance.