Ethnicity predicts viral rebound after travel to the tropics in HIV-infected travelers to the tropics in the Swiss HIV Cohort Study.

OBJECTIVES: The number of HIV-infected individuals from developed countries travelling to tropical and subtropical areas has increased as a result of the clinical and survival benefits of combination antiretroviral therapy. The aim of our study was to describe the traveler population in the SHCS and to determine the frequency of viral rebound in virologically suppressed individuals after a travel episode to the tropics compared to non-travelers. METHODS: Swiss HIV Cohort Study participants with at least one follow-up visit between 1 January 1989 and 28 February 2015 were eligible for inclusion in the study. The primary outcome was the occurrence of viral rebound (viral load > 200 HIV-1 RNA copies/mL) after a travel episode compared with a nontravel episode in previously suppressed individuals (≤ 200 copies/mL). All virologically suppressed patients contributed multiple travel or nontravel episodes to the analysis. Logistic regression was performed including factors associated with viral rebound. RESULTS: We included 16 635 patients in the study, of whom 6084 (36.5%) had ever travelled to the tropics. Travel frequency increased over time, with travellers showing better HIV parameters than nontravellers [less advanced Centers for Disease Control and Prevention (CDC) stage and higher CD4 count nadir]. Viral rebound was seen in 477 (3.9%) of 12 265 travel episodes and in 5121 (4.5%) of 114 884 nontravel episodes [unadjusted odds ratio (OR) 0.87; 95% confidence interval (CI) 0.78-0.97]. Among these 477 post-travel viral rebounds, 115 had a resistance test performed and 51 (44%) of these showed new resistance mutations. Compared with European and North American patients, the odds for viral rebound were significantly lower in Southeast Asian (OR 0.67; 95% CI 0.51-0.88) and higher in sub-Saharan African (SSA) patients (OR 1.41; 95% CI 1.22-1.62). Travel further increased the odds of viral rebound in SSA patients (OR 2.00; 95% CI 1.53-2.61). CONCLUSIONS: Region of origin is the main risk factor for viral rebound rather than travel per se. Pre-travel adherence counselling should focus on patients of SSA origin.

Postnatal retention in HIV care: insight from the Swiss HIV Cohort Study over a 15-year observational period.

OBJECTIVES: The aim of this study was to quantify loss to follow-up (LTFU) in HIV care after delivery and to identify risk factors for LTFU, and implications for HIV disease progression and subsequent pregnancies. METHODS: We used data on pregnancies within the Swiss HIV Cohort Study from 1996 to 2011. A delayed clinical visit was defined as > 180 days and LTFU as no visit for > 365 days after delivery. Logistic regression analysis was used to identify risk factors for LTFU. RESULTS: A total of 695 pregnancies in 580 women were included in the study, of which 115 (17%) were subsequent pregnancies. Median maternal age was 32 years (IQR 28-36 years) and 104 (15%) women reported any history of injecting drug use (IDU). Overall, 233 of 695 (34%) women had a delayed visit in the year after delivery and 84 (12%) women were lost to follow-up. Being lost to follow-up was significantly associated with a history of IDU [adjusted odds ratio (aOR) 2.79; 95% confidence interval (CI) 1.32-5.88; P = 0.007] and not achieving an undetectable HIV viral load (VL) at delivery (aOR 2.42; 95% CI 1.21-4.85; P = 0.017) after adjusting for maternal age, ethnicity and being on antiretroviral therapy (ART) at conception. Forty-three of 84 (55%) women returned to care after LTFU. Half of them (20 of 41) with available CD4 had a CD4 count < 350 cells/µL and 15% (six of 41) a CD4 count < 200 cells/µL at their return. CONCLUSIONS: A history of IDU and detectable HIV VL at delivery were associated with LTFU. Effective strategies are warranted to retain women in care beyond pregnancy and to avoid CD4 cell count decline. ART continuation should be advised especially if a subsequent pregnancy is planned.

An evolutionary hot spot: the pNGR234b replicon of Rhizobium sp. strain NGR234.

Rhizobium sp. strain NGR234 has an exceptionally broad host range and is able to nodulate more than 112 genera of legumes. Since the overall organization of the NGR234 genome is strikingly similar to that of the narrow-host-range symbiont Rhizobium meliloti strain 1021 (also known as Sinorhizobium meliloti), the obvious question is why are the spectra of hosts so different? Study of the early symbiotic genes of both bacteria (carried by the SymA plasmids) did not provide obvious answers. Yet, both rhizobia also possess second megaplasmids that bear, among many other genes, those that are involved in the synthesis of extracellular polysaccharides (EPSs). EPSs are involved in fine-tuning symbiotic interactions and thus may help answer the broad- versus narrow-host-range question. Accordingly, we sequenced two fragments (total, 594 kb) that encode 575 open reading frames (ORFs). Comparisons revealed 19 conserved gene clusters with high similarity to R. meliloti, suggesting that a minimum of 28% (158 ORFs) of the genetic information may have been acquired from a common ancestor. The largest conserved cluster carried the exo and exs genes and contained 31 ORFs. In addition, nine highly conserved regions with high similarity to Agrobacterium tumefaciens C58, Bradyrhizobium japonicum USDA110, and Mesorhizobium loti strain MAFF303099, as well as two conserved clusters that are highly homologous to similar regions in the plant pathogen Erwinia carotovora, were identified. Altogether, these findings suggest that >/==" BORDER="0">40% of the pNGR234b genes are not strain specific and were probably acquired from a wide variety of other microbes. The presence of 26 ORFs coding for transposases and site-specific integrases supports this contention. Surprisingly, several genes involved in the degradation of aromatic carbon sources and genes coding for a type IV pilus were also found.

Medicago truncatula plants overexpressing the early nodulin gene enod40 exhibit accelerated mycorrhizal colonization and enhanced formation of arbuscules.

The mutualistic symbiosis between flowering plants and arbuscular mycorrhizal fungi is extremely abundant in terrestrial ecosystems. In this symbiosis, obligately biotrophic fungi colonize the root of the host plants, which can benefit from these fungi by enhanced access to mineral nutrients in the soil, especially phosphorus. One of the main goals of research on this symbiosis is to find plant genes that control fungal development in the host plant. In this work, we show that mycorrhizal colonization is regulated by enod40, an early nodulin gene known to be involved in the nodule symbiosis of legumes with nitrogen-fixing bacteria. Medicago truncatula plants overexpressing enod40 exhibited stimulated mycorrhizal colonization in comparison with control plants. Overexpression of enod40 promoted fungal growth in the root cortex and increased the frequency of arbuscule formation. Transgenic lines with suppressed levels of enod40 transcripts, likely via a cosuppression phenomenon induced by the transgene, exhibited reduced mycorrhizal colonization. Hence, enod40 might be a plant regulatory gene involved in the control of the mycorrhizal symbiosis.

Substrate specificity and antifungal activity of recombinant tobacco class I chitinases.

Endochitinases contribute to the defence response of plants against chitin-containing pathogens. The vacuolar class I chitinases consist of an N-terminal cysteine-rich domain (CRD) linked by a glycine-threonine-rich spacer with 4-hydroxylated prolyl residues to the catalytic domain. We examined the functional role of the CRD and spacer region in class I chitinases by comparing wild-type chitinase A (CHN A) of Nicotiana tabacum with informative recombinant forms. The chitinases were expressed in transgenic N. sylvestris plants, purified to near homogeneity, and their structures confirmed by mass spectrometry and partial sequencing. The enzymes were tested for their substrate preference towards chitin, lipo-chitooligosaccharide Nod factors of Rhizobium, and bacterial peptidoglycans (lysozyme activity) as well as for their capacity to inhibit hyphal growth of Trichoderma viride. Deletion of the CRD and spacer alone or in combination resulted in a modest <50% reduction of hydrolytic activity relative to CHN A using colloidal chitin or M. lysodeikticus walls as substrates; whereas, antifungal activity was reduced by up to 80%. Relative to CHN A, a variant with two spacers in tandem, which binds chitin, showed very low hydrolytic activity towards chitin and Nod factors, but comparable lysozyme activity and enhanced antifungal activity. Neither hydrolytic activity, substrate specificity nor antifungal activity were strictly correlated with the CRD-mediated capacity to bind chitin. This suggests that the presence of the chitin-binding domain does not have a major influence on the functions of CHN A examined. Moreover, the results with the tandem-spacer variant raise the possibility that substantial chitinolytic activity is not essential for inhibition of T. viride growth by CHN A.

Nod factors and chitooligomers elicit an increase in cytosolic calcium in aequorin-expressing soybean cells.

Rhizobial Nod factors (NFs) function as nodulation signals that trigger symbiotic responses of leguminous host plants. NFs consist of a chitin oligomer backbone carrying a fatty acid at the non-reducing end. Depending on the rhizobial strain, NFs carry additional substituents, which may determine host specificity. Transgenic suspension-cultured soybean (Glycine max [L.] Merr.) cells expressing aequorin have been used to record cytosolic [Ca(2+)] changes upon treatment with purified NFs and chitin fragments. Both compounds elicited an increase of cytosolic [Ca(2+)] at nanomolar concentrations. The shape and amplitude of cytosolic [Ca(2+)] changes was similar to the response elicited by un-derivatized chitin oligomers. Cells challenged first with NFs did not respond to a subsequent treatment with chitin oligomers and vice versa. Dose-response experiments showed that un-derivatized chitin oligomers were more active compared with NFs. The capacity of NFs to elicit the calcium response depended on their structure. The presence of reducing end substituents in methylfucosylated NFs from Rhizobium sp. NGR234 and the O-acetyl group at the non-reducing end in NFs from Sinorhizobium meliloti attenuated the activity to cause the calcium changes. The sulfate group in NFs from Rhizobium tropici did not affect the elicitor activity. Pentameric S. meliloti NFs were more active than tetrameric molecules, whereas trimeric or dimeric degradation products were inactive. Substituents in NFs may have the function to avoid stimulation of defense reactions mediated by the perception system for chitin oligomers.

Nod factors of Rhizobium leguminosarum bv. viciae and their fucosylated derivatives stimulate a nod factor cleaving activity in pea roots and are hydrolyzed in vitro by plant chitinases at different rates.

Nod factors (NFs) are rhizobial lipo-chitooligosaccharide signals that trigger root nodule development in legumes. Modifications of NF structures influence their biological activity and affect their degradation by plant chitinases. Nodulation of certain pea cultivars by Rhizobium leguminosarum bv. viciae requires modification of NFs at the reducing end by either an O-acetyl or a fucosyl group. Fucosylated NFs were produced by an in vitro reaction with NodZ fucosyltransferase and purified. Their biological activity on pea was tested by measuring their capacity to stimulate the activity of a hydrolase that cleaves NFs. Nonmodified and fucosylated NFs displayed this activity at nano- to picomolar concentrations, while a sulfated NF from Sinorhizobium meliloti was inactive. In an additional series of experiments, the stability of non-modified and fucosylated NFs in the presence of purified tobacco chitinases was compared. The presence of the fucosyl group affected the degradation rates and the accessibility of specific cleavage sites on the chitooligosaccharide backbone. These results suggest that the fucosyl group in NFs also weakens the interaction of NFs with certain chitinases or chitinase-related proteins in pea roots.

Molecular basis of symbiotic promiscuity.

Eukaryotes often form symbioses with microorganisms. Among these, associations between plants and nitrogen-fixing bacteria are responsible for the nitrogen input into various ecological niches. Plants of many different families have evolved the capacity to develop root or stem nodules with diverse genera of soil bacteria. Of these, symbioses between legumes and rhizobia (Azorhizobium, Bradyrhizobium, Mesorhizobium, and Rhizobium) are the most important from an agricultural perspective. Nitrogen-fixing nodules arise when symbiotic rhizobia penetrate their hosts in a strictly controlled and coordinated manner. Molecular codes are exchanged between the symbionts in the rhizosphere to select compatible rhizobia from pathogens. Entry into the plant is restricted to bacteria that have the "keys" to a succession of legume "doors". Some symbionts intimately associate with many different partners (and are thus promiscuous), while others are more selective and have a narrow host range. For historical reasons, narrow host range has been more intensively investigated than promiscuity. In our view, this has given a false impression of specificity in legume-Rhizobium associations. Rather, we suggest that restricted host ranges are limited to specific niches and represent specialization of widespread and more ancestral promiscuous symbioses. Here we analyze the molecular mechanisms governing symbiotic promiscuity in rhizobia and show that it is controlled by a number of molecular keys.

N-deacetylation of Sinorhizobium meliloti Nod factors increases their stability in the Medicago sativa rhizosphere and decreases their biological activity.

Nod factors excreted by rhizobia are signal molecules that consist of a chitin oligomer backbone linked with a fatty acid at the nonreducing end. Modifications of the Nod factor structures influence their stability in the rhizosphere and their biological activity. To test the function of N-acetyl groups in Nod factors, NodSm-IV(C16:2,S) from Sinorhizobium meliloti was enzymatically N-deacetylated in vitro with purified chitin deacetylase from Colletotrichum lindemuthianum. A family of partially and completely deacetylated derivatives was produced and purified. The most abundant chemical structures identified by mass spectrometry were GlcN(C16:2)-GlcNAc-GlcNH2-GlcNAc(OH)(S), GlcN(C16,2)-GlcNAc-GlcNH2-GlcNH2(OH)(S), and GlcN(C16:2)-GlcNH2-GlcNH2-GlcNH2(OH)(S). In contrast to NodSm-IV(C16:2,S), the purified N-deacetylated derivatives were stable in the rhizosphere of Medicago sativa, indicating that the N-acetyl groups make the carbohydrate moiety of Nod factors accessible for glycosyl hydrolases of the host plant. The N-deacetylated derivatives displayed only a low level of activity in inducing root hair deformation. Furthermore, the N-deacetylated molecules were not able to stimulate Nod factor degradation by M. sativa roots, a response elicited by active Nod factors. These data show that N-acetyl groups of Nod factors are required for biological activity.

Low chitinase activity in Acacia myrmecophytes: a potential trade-off between biotic and chemical defences?

We determined chitinase activity in leaves of four myrmecophytic and four non-myrmecophytic leguminous species at the plants' natural growing sites in Mexico. Myrmecophytic plants (or 'ant plants') have obligate mutualisms with ants protecting them against herbivores and pathogenic fungi. Plant chitinases can be considered a reliable measure of plant resistance to pathogenic fungi. The myrmecophytic Acacia species, which were colonised by mutualistic ants, exhibited at least six-fold lower levels of chitinase activity compared with the non-myrmecophytic Acacia farnesiana and three other non-myrmecophytes. Though belonging to different phylogenetic groups, the myrmecophytic Acacia species formed one distinct group in the data set, which was clearly separated from the non-myrmecophytic species. These findings allowed for comparison between two recent hypotheses that attempt to explain low chitinase activity in ant plants. Most probably, chitinases are reduced in myrmecophytic plant species because these are effectively defended indirectly due to their symbiosis with mutualistic ants.

Purification and characterization of a novel chitinase-lysozyme, of another chitinase, both hydrolysing Rhizobium meliloti Nod factors, and of a pathogenesis-related protein from Medicago sativa roots.

The symbiosis between Rhizobium meliloti and Medicago sativa (Leguminosae) involves the interaction of lipochito-oligosaccharides (Nod factors) excreted by bacteria with specific proteins of the host plant. The cleavage of Nod factors can be used as an enzymic assay to identify novel hydrolytic enzymes. Here a soluble extract of 3-day-old roots was fractionated by anion exchange, affinity chromatography, gel filtration and native electrophoresis. Two acidic chitinases (pI 4.6-5.4), CHIT24 and CHIT36, designated in accordance with their molecular mass in kDa, were separated. CHIT24 cleaves all tested Nod factors to produce lipotrisaccharides with the preference NodRm-V(S)>NodRm-IV >NodRm-IV(S)>=NodRm-IV(Ac,S); it also hydrolyses colloidal 3H-chitin and has lysozyme activity. The kinetics of Nod factor degradation by CHIT24 depends on substrate structural parameters, namely the length of the oligosaccharide chain and sulphation (S) at the reducing end, but not much on acetylation (Ac) at the non-reducing end. The 25-residue N-terminal sequence of CHIT24 has no similarity with known chitinases or lysozymes, indicating that it is a novel type of hydrolase. CHIT36 also hydrolyses NodRm-V(S) into NodRm-III, but it is inactive towards NodRm-IV(S) and NodRm-IV(Ac,S) formed by R. meliloti. Finally, a 17 kDa protein, P17, was co-purified with CHIT24. It neither degrades Nod factors nor exhibits lysozyme activity and shows complete identity, at the 15-residue N-terminal sequence, with a class 10 pathogenesis-related protein, PR-10.

nodD2 of Rhizobium sp. NGR234 is involved in the repression of the nodABC operon.

Transcriptional regulators of the lysR family largely control the expression of bacterial symbiotic genes. Rhizobium sp. NGR234 contains at least four members of this family: two resemble nodD, while two others are more closely related to syrM. Part of the extremely broad host range of NGR234 can be attributed to nodD1, although the second gene shares a high degree of DNA sequence homology with nodD2 of R. fredii USDA191. A nodD2 mutant of NGR234 was constructed by insertional mutagenesis. This mutant (NGR omega nodD2) was deficient in nitrogen fixation on Vigna unguiculata and induced pseudonodules on Tephrosia vogelii. Several other host plants were tested, but no correlation could be drawn between the phenotype and nodule morphology. Moreover, nodD2 has a negative effect on the production of Nod factors: mutation of this gene results in a fivefold increase in Nod factor production. Surprisingly, while the structure of Nod factors from free-living cultures of NGR omega nodD2 remained unchanged, those from V. unguiculata nodules induced by the same strain are non-fucosylated and have a lower degree of oligomerization. In other words, developmental regulation of Nod factor production is also abolished in this mutant. Competitive RNA hybridizations, gene fusions and mobility shift assays confirmed that nodD2 downregulates expression of the nodABC operon.

Rhizobial Nodulation Factors Stimulate Mycorrhizal Colonization of Nodulating and Nonnodulating Soybeans.

Legumes form tripartite symbiotic associations with noduleinducing rhizobia and vesicular-arbuscular mycorrhizal fungi. Co-inoculation of soybean (Glycine max [L.] Merr.) roots with Bradyrhizobium japonicum 61-A-101 considerably enhanced colonization by the mycorrhizal fungus Glomus mosseae. A similar stimulatory effect on mycorrhizal colonization was also observed in nonnodulating soybean mutants when inoculated with Bradyrhizobium japonicum and in wild-type soybean plants when inoculated with ineffective rhizobial strains, indicating that a functional rhizobial symbiosis is not necessary for enhanced mycorrhiza formation. Inoculation with the mutant Rhizobium sp. NGR[delta]nodABC, unable to produce nodulation (Nod) factors, did not show any effect on mycorrhiza. Highly purified Nod factors also increased the degree of mycorrhizal colonization. Nod factors from Rhizobium sp. NGR234 differed in their potential to promote fungal colonization. The acetylated factor NodNGR-V (MeFuc, Ac), added at concentrations as low as 10-9 M, was active, whereas the sulfated factor, NodNGR-V (MeFuc, S), was inactive. Several soybean flavonoids known to accumulate in response to the acetylated Nod factor showed a similar promoting effect on mycorrhiza. These results suggest that plant flavonoids mediate the Nod factor-induced stimulation of mycorrhizal colonization in soybean roots.

Lipo-chitooligosaccharide Nodulation Signals from Rhizobium meliloti Induce Their Rapid Degradation by the Host Plant Alfalfa.

Extracellular enzymes from alfalfa (Medicago sativa L.) involved in the degradation of nodulation (Nod) factors could be distinguished by their different cleavage specificities and were separated by lectin affinity chromatography. A particular glycoprotein was able to release an acylated lipo-disaccharide from all tested Nod factors having an oligosaccharide chain length of four or five residues. Structural modifications of the basic lipo-chitooligosaccharide did not affect the cleavage site and had only weak influence on the cleavage efficiency of Nod factors tested. The acylated lipo-trisaccharide was resistant to degradation. When alfalfa roots were preincubated with Nod factors at nanomolar concentrations, the activity of the dimer-forming enzyme was stimulated up to 6-fold within a few hours. The inducing activity of Nod factors decreased in the order NodRm-IV(C16:2,Ac,S) > NodRm-IV(C16:2,S) and NodRm-V(C16:2,Ac,S) > NodRm-V(C16:2,S) > NodRm-IV(C16:0,S) > NodRm-IV(C16:2). Pretreatment with NodRm-III(C16:2) as well as unmodified chitooligosaccharides did not stimulate the dimer-forming enzyme. Roots preincubated with Rhizobium meliloti showed similar stimulation of the dimer-forming activity. Mutant strains unable to produce Nod factors did not enhance the hydrolytic activity. These results indicate a rapid feedback inactivation of Nod signals after their perception by the host plant alfalfa.

In vitro sulfotransferase activity of Rhizobium meliloti NodH protein: lipochitooligosaccharide nodulation signals are sulfated after synthesis of the core structure.

The Rhizobium common nod gene products NodABC are involved in the synthesis of the core lipochitooligosaccharide (Nod factor) structure, whereas the products of the host-specific nod genes are necessary for diverse structural modifications, which vary in different Rhizobium species. The sulfate group attached to the Rhizobium meliloti Nod signal is necessary for activity on the host plant alfalfa, while its absence renders the Nod factor active on the non-host plant vetch. This substituent is therefore a major determinant of host specificity. The exact biosynthetic pathway of Nod factors has not been fully elucidated. In particular, it is not known why some chemical modifications are introduced with high fidelity whereas others are inaccurate, giving rise to a family of different Nod factor structures produced by a single Rhizobium strain. Using protein extracts and partially purified recombinant NodH protein obtained from Escherichia coli expressing the R. meliloti nodH gene, we demonstrate here NodH-dependent in vitro sulfotransferase activity. Kinetic analyses with Nod factors, chitooligosaccharides, and their deacetylated derivatives revealed that Nod factors are the preferred substrate for the sulfate transfer. Moreover, the tetrameric Nod factor, NodRm-IV, was a better substrate than the trimer, NodRm-III, or the pentamer, NodRm-V. These data suggest that the core lipochitooligosaccharide structure must be synthesized prior to its host-specific modification with a sulfate group. Since in R. meliloti tetrameric Nod factors are the most abundant and the most active ones, high affinity of NodH for the appropriate tetrameric substrate guarantees its modification and thus contributes to the fidelity of host-specific behavior.

Perception of Rhizobium nodulation factors by tomato cells and inactivation by root chitinases.

The bacterial genera Rhizobium and Bradyrhizobium, nitrogen-fixing symbionts of legumes, secrete specific lipo-chitooligosaccharides that induce the formation of nodules on their host plants. When preparations of such nodulation-inducing factors (Nod factors) were added to suspension-cultured tomato cells, a rapid and transient alkalinization of the culture medium occurred. Lipo-oligosaccharide preparations from Rhizobium or Bradyrhizobium treated with flavonoids, known inducers of Nod factor synthesis, were up to 100 times more potent in inducing alkalinization than the ones from untreated bacteria. The activity was absent from preparations of the mutant strain Rhizobium sp. NGR234 delta nodABC, unable to produce any Nod factors. Preparations of Nod factors from various bacteria as well as individual, highly purified Nod factors from Rhizobium sp. NGR(pA28) induced alkalinization in the tomato cell cultures at nanomolar concentrations. This demonstrates that Nod factors can be perceived by tomato, a nonhost of rhizobia. Using the alkalinization response as a sensitive bioassay, Nod factors were found to be inactivated by plant chitinases. Root chitinases purified from different legumes differed in their potential to inactivate differently substituted Nod factors produced by Rhizobium sp. NGR(pA28). This indicates that the specificity of the bacterium-host plant interaction may be due, at least in part, to differential inactivation of Nod factors by root chitinases.

Chitinase and peroxidase in effective (fix(+)) and ineffective (fix (-)) soybean nodules.

Chitinase and peroxidase, two enzymes thought to be involved in the defense of plants against pathogens, were measured in soybean (Glycine max L. Merr.) roots and in nodules colonized by Bradyrhizobium japonicum strains differing in their symbiotic potential. Activities of both enzymes were higher in nodules than in roots. In "effective", nitrogen-fixing nodules, colonized by wild-type bacteria, chitinase and peroxidase activities had low levels in the central infected zone and were enhanced primarily in the nodule cortex. An ascorbate-specific peroxidase, possibly involved in radical scavenging, had similarly high activities in the infected zone and in the cortex. "Ineffective" nodules colonized by bacteria unable to fix nitrogen symbiotically showed a similar distribution of chitinase and peroxidase. In another type of "ineffective" nodule, colonized by a B. japonicum strain eliciting a hypersensitive response, activities of both enzymes were enhanced to a similar degree in the infected zone as well as in the cortex. Tissue prints using a direct assay for peroxidase and an antiserum against bean chitinase corroborated these results. The antiserum against bean chitinase cross-reacted with a nodule protein of Mr 32 000; it inhibited most of the chitinase activity in the nodules but barely affected the chitinase in uninfected roots. It is concluded that proteins characteristic of the defense reaction accumulate in the cortex of nodules independently of their ability to fix nitrogen, and in the entire body of hypersensitively reacting nodules.