Comparative secretome analysis of Striga and Cuscuta species identifies candidate virulence factors for two evolutionarily independent parasitic plant lineages.

BACKGROUND: Many parasitic plants of the genera Striga and Cuscuta inflict huge agricultural damage worldwide. To form and maintain a connection with a host plant, parasitic plants deploy virulence factors (VFs) that interact with host biology. They possess a secretome that represents the complement of proteins secreted from cells and like other plant parasites such as fungi, bacteria or nematodes, some secreted proteins represent VFs crucial to successful host colonisation. Understanding the genome-wide complement of putative secreted proteins from parasitic plants, and their expression during host invasion, will advance understanding of virulence mechanisms used by parasitic plants to suppress/evade host immune responses and to establish and maintain a parasite-host interaction. RESULTS: We conducted a comparative analysis of the secretomes of root (Striga spp.) and shoot (Cuscuta spp.) parasitic plants, to enable prediction of candidate VFs. Using orthogroup clustering and protein domain analyses we identified gene families/functional annotations common to both Striga and Cuscuta species that were not present in their closest non-parasitic relatives (e.g. strictosidine synthase like enzymes), or specific to either the Striga or Cuscuta secretomes. For example, Striga secretomes were strongly associated with 'PAR1' protein domains. These were rare in the Cuscuta secretomes but an abundance of 'GMC oxidoreductase' domains were found, that were not present in the Striga secretomes. We then conducted transcriptional profiling of genes encoding putatively secreted proteins for the most agriculturally damaging root parasitic weed of cereals, S. hermonthica. A significant portion of the Striga-specific secretome set was differentially expressed during parasitism, which we probed further to identify genes following a 'wave-like' expression pattern peaking in the early penetration stage of infection. We identified 39 genes encoding putative VFs with functions such as cell wall modification, immune suppression, protease, kinase, or peroxidase activities, that are excellent candidates for future functional studies. CONCLUSIONS: Our study represents a comprehensive secretome analysis among parasitic plants and revealed both similarities and differences in candidate VFs between Striga and Cuscuta species. This knowledge is crucial for the development of new management strategies and delaying the evolution of virulence in parasitic weeds.

Genome-enabled discovery of candidate virulence loci in Striga hermonthica, a devastating parasite of African cereal crops.

Parasites have evolved proteins, virulence factors (VFs), that facilitate plant colonisation, however VFs mediating parasitic plant-host interactions are poorly understood. Striga hermonthica is an obligate, root-parasitic plant of cereal hosts in sub-Saharan Africa, causing devastating yield losses. Understanding the molecular nature and allelic variation of VFs in S. hermonthica is essential for breeding resistance and delaying the evolution of parasite virulence. We assembled the S. hermonthica genome and identified secreted proteins using in silico prediction. Pooled sequencing of parasites growing on a susceptible and a strongly resistant rice host allowed us to scan for loci where selection imposed by the resistant host had elevated the frequency of alleles contributing to successful colonisation. Thirty-eight putatively secreted VFs had very different allele frequencies with functions including host cell wall modification, protease or protease inhibitor and kinase activities. These candidate loci had significantly higher Tajima's D than the genomic background, consistent with balancing selection. Our results reveal diverse strategies used by S. hermonthica to overcome different layers of host resistance. Understanding the maintenance of variation at virulence loci by balancing selection will be critical to managing the evolution of virulence as part of a sustainable control strategy.

SMAX1-dependent seed germination bypasses GA signalling in Arabidopsis and Striga.

Parasitic plant infestations dramatically reduce the yield of many major food crops of sub-Saharan Africa and pose a serious threat to food security on that continent1. The first committed step of a successful infestation is the germination of parasite seeds primarily in response to a group of related small-molecule hormones called strigolactones (SLs), which are emitted by host roots2. Despite the important role of SLs, it is not clear how host-derived SLs germinate parasitic plants. In contrast, gibberellins (GA) acts as the dominant hormone for stimulation of germination in non-parasitic plant species by inhibiting a set of DELLA repressors3. Here, we show that expression of SL receptors from the parasitic plant Striga hermonthica in the presence of SLs circumvents the GA requirement for germination of Arabidopsis thaliana seed. Striga receptors co-opt and enhance signalling through the HYPOSENSITIVE TO LIGHT/KARRIKIN INSENSITIVE 2 (AtHTL/KAI2) pathway, which normally plays a rudimentary role in Arabidopsis seed germination4,5. AtHTL/KAI2 negatively controls the SUPPRESSOR OF MAX2 1 (SMAX1) protein5, and loss of SMAX1 function allows germination in the presence of DELLA repressors. Our data suggest that ligand-dependent inactivation of SMAX1 in Striga and Arabidopsis can bypass GA-dependent germination in these species.

Publisher Correction: Farming with crops and rocks to address global climate, food and soil security.

In the version of this Perspective originally published, 'acidification' was incorrectly spelt as 'adification' in Fig. 4. This has now been corrected.

Farming with crops and rocks to address global climate, food and soil security.

The magnitude of future climate change could be moderated by immediately reducing the amount of CO2 entering the atmosphere as a result of energy generation and by adopting strategies that actively remove CO2 from it. Biogeochemical improvement of soils by adding crushed, fast-reacting silicate rocks to croplands is one such CO2-removal strategy. This approach has the potential to improve crop production, increase protection from pests and diseases, and restore soil fertility and structure. Managed croplands worldwide are already equipped for frequent rock dust additions to soils, making rapid adoption at scale feasible, and the potential benefits could generate financial incentives for widespread adoption in the agricultural sector. However, there are still obstacles to be surmounted. Audited field-scale assessments of the efficacy of CO2 capture are urgently required together with detailed environmental monitoring. A cost-effective way to meet the rock requirements for CO2 removal must be found, possibly involving the recycling of silicate waste materials. Finally, issues of public perception, trust and acceptance must also be addressed.

Genetic variation and host-parasite specificity of Striga resistance and tolerance in rice: the need for predictive breeding.

The parasitic weeds Striga asiatica and Striga hermonthica cause devastating yield losses to upland rice in Africa. Little is known about genetic variation in host resistance and tolerance across rice genotypes, in relation to virulence differences across Striga species and ecotypes. Diverse rice genotypes were phenotyped for the above traits in S. asiatica- (Tanzania) and S. hermonthica-infested fields (Kenya and Uganda) and under controlled conditions. New rice genotypes with either ecotype-specific or broad-spectrum resistance were identified. Resistance identified in the field was confirmed under controlled conditions, providing evidence that resistance was largely genetically determined. Striga-resistant genotypes contributed to yield security under Striga-infested conditions, although grain yield was also determined by the genotype-specific yield potential and tolerance. Tolerance, the physiological mechanism mitigating Striga effects on host growth and physiology, was unrelated to resistance, implying that any combination of high, medium or low levels of these traits can be found across rice genotypes. Striga virulence varies across species and ecotypes. The extent of Striga-induced host damage results from the interaction between parasite virulence and genetically determined levels of host-plant resistance and tolerance. These novel findings support the need for predictive breeding strategies based on knowledge of host resistance and parasite virulence.

Do NERICA rice cultivars express resistance to Striga hermonthica (Del.) Benth. and Striga asiatica (L.) Kuntze under field conditions?

The parasitic weeds Striga asiatica and Striga hermonthica cause high yield losses in rain-fed upland rice in Africa. Two resistance classes (pre- and post-attachment) and several resistant genotypes have been identified among NERICA (New Rice for Africa) cultivars under laboratory conditions (in vitro) previously. However, little is known about expression of this resistance under field conditions. Here we investigated (1) whether resistance exhibited under controlled conditions would express under representative Striga-infested field conditions, and (2) whether NERICA cultivars would achieve relatively good grain yields under Striga-infested conditions. Twenty-five rice cultivars, including all 18 upland NERICA cultivars, were screened in S. asiatica-infested (in Tanzania) and S. hermonthica-infested (in Kenya) fields during two seasons. Additionally, a selection of cultivars was tested in vitro, in mini-rhizotron systems. For the first time, resistance observed under controlled conditions was confirmed in the field for NERICA-2, -5, -10 and -17 (against S. asiatica) and NERICA-1 to -5, -10, -12, -13 and -17 (against S. hermonthica). Despite high Striga-infestation levels, yields of around 1.8 t ha-1 were obtained with NERICA-1, -9 and -10 (in the S. asiatica-infested field) and around 1.4 t ha-1 with NERICA-3, -4, -8, -12 and -13 (in the S. hermonthica-infested field). In addition, potential levels of tolerance were identified in vitro, in NERICA-1, -17 and -9 (S. asiatica) and in NERICA-1, -17 and -10 (S. hermonthica). These findings are highly relevant to rice agronomists and breeders and molecular geneticists working on Striga resistance. In addition, cultivars combining broad-spectrum resistance with good grain yields in Striga-infested fields can be recommended to rice farmers in Striga-prone areas.

Diversity of planktonic and attached bacterial communities in a phenol-contaminated sandstone aquifer.

Polluted aquifers contain indigenous microbial communities with the potential for in situ bioremediation. However, the effect of hydrogeochemical gradients on in situ microbial communities (especially at the plume fringe, where natural attenuation is higher) is still not clear. In this study, we used culture-independent techniques to investigate the diversity of in situ planktonic and attached bacterial communities in a phenol-contaminated sandstone aquifer. Within the upper and lower plume fringes, denaturing gradient gel electrophoresis profiles indicated that planktonic community structure was influenced by the steep hydrogeochemical gradient of the plume rather than the spatial location in the aquifer. Under the same hydrogeochemical conditions (in the lower plume fringe, 30 m below ground level), 16S rRNA gene cloning and sequencing showed that planktonic and attached bacterial communities differed markedly and that the attached community was more diverse. The 16S rRNA gene phylogeny also suggested that a phylogenetically diverse bacterial community operated at this depth (30 mbgl), with biodegradation of phenolic compounds by nitrate-reducing Azoarcus and Acidovorax strains potentially being an important process. The presence of acetogenic and sulphate-reducing bacteria only in the planktonic clone library indicates that some natural attenuation processes may occur preferentially in one of the two growth phases (attached or planktonic). Therefore, this study has provided a better understanding of the microbial ecology of this phenol-contaminated aquifer, and it highlights the need for investigating both planktonic and attached microbial communities when assessing the potential for natural attenuation in contaminated aquifers.

Gall formation in clubroot-infected Arabidopsis results from an increase in existing meristematic activities of the host but is not essential for the completion of the pathogen life cycle.

Plasmodiophora brassicae (clubroot) infection leads to reprogramming of host development resulting in the formation of characteristic galls. In this work we explored the cellular events that underly gall formation in Arabidopsis thaliana with the help of molecular markers of cell division (CYCB1:GUS) and meristematic activity (ANT:GUS). Our results show that gall development involved the amplification of existing meristematic activities within the vascular cambium (VC) and phloem parenchyma (PP) cells in the region of the hypocotyl. Additionally we found that the increase in VC activity and prolonged maintenance of cambial-derived cells in a meristematic state was crucial for gall formation; disruption of the VC activity significantly decreased the gall size. Gall formation also perturbed vascular development with a significant reduction in xylem and increase in PP in infected plants. This situation was reflected in a decrease in transcripts of key factors promoting xylogenesis (VND6, VND7 and MYB46) and an increase in those promoting phloem formation and function (APL, SUC2). Finally we show, using the cell cycle inhibitor ICK1/KRP1 and a cle41 mutant with altered regulation of cambial stem cell maintenance and differentiation, that a decrease in gall formation did not prevent pathogen development. This finding demonstrates that although gall formation is a typical symptom of the disease and influences numbers of spores produced, it is not required for completion of the pathogen life cycle. Together, these results provide an insight into the relationship of the cellular events that accompany Plasmodiophora infection and their role in disease progression.

Impacts of logging on density-dependent predation of dipterocarp seeds in a South East Asian rainforest.

Much of the forest remaining in South East Asia has been selectively logged. The processes promoting species coexistence may be the key to the recovery and maintenance of diversity in these forests. One such process is the Janzen-Connell mechanism, where specialized natural enemies such as seed predators maintain diversity by inhibiting regeneration near conspecifics. In Neotropical forests, anthropogenic disturbance can disrupt the Janzen-Connell mechanism, but similar data are unavailable for South East Asia. We investigated the effects of conspecific density (two spatial scales) and distance from fruiting trees on seed and seedling survival of the canopy tree Parashorea malaanonan in unlogged and logged forests in Sabah, Malaysia. The production of mature seeds was higher in unlogged forest, perhaps because high adult densities facilitate pollination or satiate pre-dispersal predators. In both forest types, post-dispersal survival was reduced by small-scale (1 m(2)) conspecific density, but not by proximity to the nearest fruiting tree. Large-scale conspecific density (seeds per fruiting tree) reduced predation, probably by satiating predators. Higher seed production in unlogged forest, in combination with slightly higher survival, meant that recruitment was almost entirely limited to unlogged forest. Thus, while logging might not affect the Janzen-Connell mechanism at this site, it may influence the recruitment of particular species.

New Rice for Africa (NERICA) cultivars exhibit different levels of post-attachment resistance against the parasitic weeds Striga hermonthica and Striga asiatica.

Striga hermonthica and S. asiatica are root parasitic weeds that infect the major cereal crops of sub-Saharan Africa causing severe losses in yield. The interspecific upland NEw RICe for Africa (NERICA) cultivars are popular amongst subsistence farmers, but little is known about their post-attachment resistance against Striga. Here, we evaluate the post-attachment resistance levels of the NERICA cultivars and their parents against ecotypes of S. hermonthica and S.asiatica, characterize the phenotype of the resistance mechanisms and determine the effect of Striga on host biomass. Some NERICA cultivars showed good broad-spectrum resistance against several Striga ecotypes, whereas others showed intermediate resistance or were very susceptible. The phenotype of a resistant interaction was often characterized by an inability of the parasite to penetrate the endodermis. Moreover, some parasites formed only a few connections to the host xylem, grew slowly and remained small. The most resistant NERICA cultivars were least damaged by Striga, although even a small number of parasites caused a reduction in above-ground host biomass. The elucidation of the molecular genetic basis of the resistance mechanisms and tolerance would allow the development of cultivars with multiple, durable resistance for use in farmers' fields.

Host plant resistance to parasitic weeds; recent progress and bottlenecks.

Parasitic witchweeds (Striga spp.) and broomrapes (Orobanche and Phelipanche spp.) directly invade the roots of crop plants connecting to the vascular system and abstracting nutrients and water. As a consequence they cause devastating losses in crop yield. Genetic resistance to parasitic weeds is a highly desirable component of any control strategy. Resistance to parasitic plants can occur at different stages of the parasite lifecycle: before attachment to the host, during penetration of the root or after establishment of vascular connections. New studies are beginning to shed light on the molecular mechanisms and signaling pathways involved in plant-plant resistance. The first resistance gene to Striga, encoding a CC-NBS-LRR Resistance protein (R) has been identified and cloned suggesting that host plants resist attack from parasitic plants using similar surveillance mechanisms as those used against fungal and bacterial pathogens. It is becoming clear that the salicylic acid (SA) signaling pathway plays an important role in resistance to parasitic plants and genes encoding pathogenesis-related (PR) proteins are upregulated in a number of the resistant interactions. New strategies for engineering resistance to parasitic plants are also being explored, including the expression of parasite-specific toxins in host roots and RNAi to silence parasite genes crucial for development.

Biofilm formation in environmental bacteria is influenced by different macromolecules depending on genus and species.

The formation of biofilms by diverse bacteria isolated from contaminated soil and groundwater on model substrata with different surface properties was assessed in a multifactorial screen. Diverse attachment phenotypes were observed as measured by crystal violet dye retention and confocal laser scanning microscopy (CLSM). Bulk measurements of cell hydrophobicity had little predictive ability in determining whether biofilms would develop on hydrophobic or hydrophilic substrata. Therefore selected pairs of bacteria from the genera Rhodococcus, Pseudomonas and Sphingomonas that exhibited different attachment phenotypes were examined in more detail using CLSM and the lipophilic dye, Nile Red. The association of Rhodococcus sp. cell membranes with lipids was shown to influence the attachment properties of these cells, but this approach was not informative for Pseudomonas and Sphingomonas sp. Confocal Raman Microspectroscopy of Rhodococcus biofilms confirmed the importance of lipids in their formation and indicated that in Pseudomonas and Sphingomonas biofilms, nucleic acids and proteins, respectively, were important in identifying the differences in attachment phenotypes of the selected strains. Treatment of biofilms with DNase I confirmed a determining role for nucleic acids as predicted for Pseudomonas. This work demonstrates that the attachment phenotypes of microbes from environmental samples to different substrata varies markedly, a diverse range of macromolecules may be involved and that these differ significantly between genera. A combination of CLSM and Raman spectroscopy distinguished between phenotypes and could be used to identify the key macromolecules involved in cell attachment to surfaces for the specific cases studied.

Evolutionary history and distance dependence control survival of dipterocarp seedlings.

One important hypothesis to explain tree-species coexistence in tropical forests suggests that increased attack by natural enemies near conspecific trees gives locally rare species a competitive advantage. Host ranges of natural enemies generally encompass several closely related plant taxa suggesting that seedlings should also do poorly around adults of closely related species. We investigated the effects of adult Parashorea malaanonan on seedling survival in a Bornean rain forest. Survival of P. malaanonan seedlings was highest at intermediate distances from parent trees while heterospecific seedlings were unaffected by distance. Leaf herbivores did not drive this relationship. Survival of seedlings was lowest for P. malaanonan, and increased with phylogenetic dissimilarity from this species, suggesting that survival of close relatives of common species is reduced. This study suggests that distance dependence contributes to species coexistence and highlights the need for further investigation into the role of shared plant enemies in community dynamics.

Dynamic changes in microbial community structure and function in phenol-degrading microcosms inoculated with cells from a contaminated aquifer.

Contamination of aquifers by organic pollutants threatens groundwater supplies and the environment. In situ biodegradation of organic pollutants by microbial communities is important for the remediation of contaminated sites, but our understanding of the relationship between microbial development and pollutant biodegradation is poor. A particular challenge is understanding the in situ status of microorganisms attached to solid surfaces, but not accessible via conventional sampling of groundwater. We have developed novel flow-through microcosms and examined dynamic changes in microbial community structure and function in a phenol-degrading system. Inoculation of these microcosms with a complex microbial community from a plume in a phenol-contaminated aquifer led to the initial establishment of a population dominated by a few species, most attached to the solid substratum. Initially, phenol biodegradation was incomplete, but as the microbial community structure became more complex, phenol biodegradation was more extensive and complete. These results were replicated between independent microcosms, indicating a deterministic succession of species. This work demonstrates the importance of examining community dynamics when assessing the potential for microbial biodegradation of organic pollutants. It provides a novel system in which such measurements can be made readily and reproducibly to study the temporal development and spatial succession of microbial communities during biodegradation of organic pollutants at interfaces within such environments.

A major QTL for resistance of rice to the parasitic plant Striga hermonthica is not dependent on genetic background.

BACKGROUND: The use of Striga-resistant germplasm is likely to be a cost-effective control strategy for preventing loss of yield owing to Striga. Previously, the authors identified quantitative trait loci (QTL) for resistance in rice to Striga hermonthica (Del.) Benth. in backcross inbred lines (BILs) derived from a cross between two cultivars Nipponbare and Kasalath. It is essential to validate QTL in different environments and/or genetic backgrounds to develop molecular markers linked to resistance QTL for use in marker-assisted selection (MAS) programmes. This study aimed to establish whether a large-effect Kasalath-derived resistance QTL allele on chromosome 4 of rice also conferred resistance in a different mapping population derived from a cross between Koshihikari and Kasalath, and to identify any further Striga resistance QTL. RESULTS: Three Striga resistance QTL were detected in Koshihikari-Kasalath BILs, two of which were derived from the Kasalath allele and one from the Koshihkari allele. The largest QTL (Kasalath allele) explained 16% of the variation in the mapping population and was located on chromosome 4. Comparison between these data and those of the authors' previous analysis revealed that the confidence intervals of the chromosome-4 QTL in the Nipponbare-Kasalath cross and the Kasalath-Koshihikari cross overlapped between 6.5 Mbp and 8 Mbp on the physical rice genome assembly. CONCLUSION: This study has both verified and narrowed down the position of a Striga resistance QTL of major effect, and demonstrated that it may be a tractable target for MAS.

Chlorophyll fluorescence imaging as tool for understanding the impact of fungal diseases on plant performance: a phenomics perspective.

Chlorophyll fluorescence imaging is a non-invasive, non-destructive means with which to examine the impact of fungal pathogens on the photosynthetic metabolism of host plants. As such, it has great potential for screening purposes in high-throughput phenomics environments. However, there is great diversity in the responses of plants to different plant-fungal pathogens and the choice of suitable experimental conditions and protocols and interpretation of the results requires both preliminary laboratory experiments and an understanding of the biology of the specific plant-pathogen interaction. In this review, we examine the interaction between biotrophic, hemi-biotrophic and necrotrophic fungal pathogens and their hosts to illustrate the extent to which chlorophyll fluorescence imaging can be used to detect the presence of disease before the appearance of visible symptoms, distinguish between compatible and incompatible fungal interactions, identify heterogeneity in photosynthetic performance within the infected leaf and provide insights into the underlying mechanisms. The limitations and challenges of using chlorophyll fluorescence imaging in high throughput screens is discussed.

Striga infestation of cereal crops - an unsolved problem in resource limited agriculture.

The parasitic weed Striga causes devastating losses in cereal yields in sub-Saharan Africa. The parasite lifecycle is intimately linked with its host via a complex interchange of signals. Understanding the molecular basis of these interactions and of host resistance to Striga is essential for the identification of genes for improving crop yield via biotechnological or marker assisted breeding strategies. Cloning and sequencing of ESTs from the 'model' parasite Triphysaria versicolor is facilitating the identification of parasitism genes. The identification of resistance to Striga in sorghum and rice germplasm is allowing molecular dissection of these traits using genomic platforms and quantitative trait loci (QTL) analysis. QTL underlying different resistance phenotypes have been identified and the use of advanced backcross populations is allowing the exploitation of sources of resistance in wild relatives of cereals.

Metabolic consequences of susceptibility and resistance (race-specific and broad-spectrum) in barley leaves challenged with powdery mildew.

In a compatible interaction biotrophic fungi often lower the yield of their hosts by reducing photosynthesis and altering the fluxes of carbon within the infected leaf. In contrast, comparatively little is known about the metabolic consequences of activating resistance responses. In this study we investigated the hypothesis that the activation of both race-specific (Mla12) and broad-spectrum (mlo) resistance pathways in barley leaves infected with Blumeria graminis represents a cost to the plant in terms of carbon production and utilization. We have shown, using quantitative imaging of chlorophyll fluorescence, that during a susceptible interaction, photosynthesis was progressively reduced both in cells directly below fungal colonies and in adjacent cells when compared with uninoculated leaves. The lower rate of photosynthesis was associated with an increase in invertase activity, an accumulation of hexoses and a down-regulation of photosynthetic gene expression. During both Mla12- and mlo-mediated resistance, photosynthesis was also reduced, most severely inhibited in cells directly associated with attempted penetration of the fungus but also in surrounding cells. These cells displayed intense autofluorescence under ultraviolet illumination indicative of the accumulation of phenolic compounds and/or callose deposition. The depression in photosynthesis was not due only to cell death but also to an alteration in source-sink relations and carbon utilization. Apoplastic (cell wall-bound) invertase activity increased more rapidly and to a much greater extent than in infected susceptible leaves and was accompanied by an accumulation of hexoses that was localized to areas of the leaf actively exhibiting resistance responses. The accumulation of hexoses was accompanied by a down-regulation in the expression of Rubisco (rbcS) and chlorophyll a/b binding protein (cab) genes (although to a lesser extent than in a compatible interaction) and with an up-regulation in the expression of the pathogenesis-related protein 1 (PR-1). These results are consistent with a role for invertase in the generation of hexoses, which may supply energy for defence reactions and/or act as signals inducing defence gene expression.

Imaging photosynthesis in wounded leaves of Arabidopsis thaliana.

Chlorophyll fluorescence imaging provides a non-invasive and non-destructive means with which to measure photosynthesis. This technique has been used, in combination with 14CO2 feeding, to study the spatial and temporal changes in source-sink relationships which occur in mechanically wounded leaves of Arabidopsis thaliana. Twenty-four hours after wounding, cells proximal to the wound margin showed a rapid induction of PhiII upon illumination (a measure of the efficiency of photosystem II photochemistry) whilst cells more distal to the wound margin exhibited a much slower induction of PhiII and a large, transient increase in NPQ (a measure of the rate constant for non-photochemical energy dissipation within the light-harvesting antenna). These results are indicative of an increase in sink strength in the vicinity of the wound and this was confirmed by the retention of 14C photosynthate in this region. It has been hypothesized that wound-induced cell wall (apoplastic) invertase (cwINV) activity plays a central role in generating localized increases in sink strength in stressed plant tissue and that hexose sugars generated by the sucrolytic activity of cwINV may act as a signal regulating gene expression. Enzyme activity measurements, quantitative RT-PCR, and T-DNA insertional mutagenesis have been used to determine that expression of AtcwINV1 is responsible for all induced cwINV activity in mechanically wounded leaves. Whilst inactivation of this gene abolished wound-induced cwINV activity, it did not affect localized alterations in source-sink relationships of wounded leaves or wound-regulated gene expression. The signals that may regulate source-sink relationships and signalling in wounded leaves are discussed.