Psidium Defenses Against Meloidogyne enterolobii: Proteomic and Microscopic Analysis of this Plant-Predator Association

Abstract

Guava (Psidium guajava), referred to as the "tropical apple," is esteemed for its sweet flavor, nutritional density, and medicinal attributes, being rich in ascorbic acid, phenolics, carotenoids, fibers, and minerals. Despite its agricultural significance, guava cultivation faces considerable challenges from plant-parasitic nematodes, particularly root-knot nematodes from the Meloidogyne spp. In South America, Meloidogyne enterolobii causes severe root damage and economic losses to this crop. Plants fight nematodes through complex immune mechanisms involving pattern recognition receptors and signaling pathways, such as pattern-triggered immunity. The present research employed comparative shotgun proteomic analysis complemented by microscopic imaging and histochemical assays of roots from susceptible P. guajava and resistant P. guineense, inoculated or not with M. enterolobii. Psidium-M. enterolobii interactions revealed intricate plant cellular responses such as giant cells formation, hypersensitivity reactions, and biochemical pathway adjustments in sucrose transport and antioxidant enzyme activities. Synthesis and accumulation of secondary metabolites like terpenes, alkaloids, and phenolics in inoculated and resistant plants were positively correlated to plant resilience. Heat shock proteins and protein disulfide isomerases also emerged as pivotal in plant response, being upregulated during nematode infection. SUMMARY: The work addresses and unravels some of the puzzle pieces in the net of processes triggered in a plant prey (Psidium spp.), of either susceptible (P. guajava) or resistant (P. guineense) phenotypes, when confronted by its nematode predator (Meloidogyne enterolobii). The main alterations detected in the roots of these plants ranged from giant cells formation, hypersensitivity reactions, biochemical adjustments in sucrose transport pathways and in antioxidant enzyme activities, to increases in secondary metabolites (terpenes, alkaloids, and phenolics) and in heat shock proteins and protein disulfide isomerases. All these defensive mechanisms were triggered by the nematode attack on both species and were more prominent in P. guineense, which positively correlates them to the plant resistance against M. enterolobii.

Keywords: crop resistance; guava (Psidium guajava); immune mechanisms; plant‐parasitic nematodes; root anatomy.

FIGURE 1

Images of the roots obtained by bright field light microscopy: GUA_20DNI (A and B); GUI_20DNI (C and D); GUA_20DAI (E and F) e; GUI_20DAI (G and H). P. guajava—GUA; P. guineense—GUI; 20 days non‐inoculated—20DNI; 20 days after inoculation—20DAI; Vascular cylinder—VC; Xylem–Xy; Phloem—Ph; Giant cells—GC; Nematode—N; Grayish‐colored cells—GCC.

FIGURE 2

Images of roots obtained by transmission electron microscopy (a)—GUA_20DNI (A and B); GUI_20DNI (C and D); GUA_20DAI (E and F); and GUI_20DAI (G and H) and scanning electron microscopy (b)—GUA_20DNI (A); GUI_20DNI (B); GUA_20DAI (C); and GUI_20DAI (D). P. guajava—GUA; P. guineense—GUI; 20 days non‐inoculated—20DNI; 20 days after inoculation—20DAI.

FIGURE 3

Images obtained by light field optical microscopy after staining for histochemical testing of Psidium roots: control roots of P. guajava (non‐inoculated with M. enterolobii)—GUA_20DNI (row 1), control roots of P. guineense (non‐inoculated)—GUI_20DNI (row 2), P. guajava roots 20 days after inoculation—GUA_20DAI (row 3) and P. guineense roots 20 days after inoculation—GUI_20DAI (row 4): control (column 1); phenolic compounds stained with ferric chloride (column 2); terpenes stained with NADI solution (column 3); and alkaloids stained with Wagner's Reagent (column 4).

FIGURE 4

Multivariate analysis of principal components for the set of 416 selected proteins from roots of Psidium. (a) [P. guajava 5 days after inoculation‐ GUA_5DAI; P. guajava 20 days after inoculation—GUA_20DAI; P. guajava after 20 days non‐inoculation of M. enterolobii—GUA_20DNI; P. guineense 5 days after inoculation—GUI_5DAI; P. guineense 20 days after inoculation—GUI_20DAI; P. guineense after 20 days non‐inoculation—GUI_20DNI], Volcano distribution plot of the up‐ and down‐regulated proteins among the whole 416 protein set from P. guajava and P. guineense samples (b) [(A) GUA_20DAI / GUA_20DNI—comparison between P. guajava 20 days after inoculation and P. guajava 20 days after non‐inoculation with M. enterolobii; (B) GUI_20DAI/GUI_20DNI—comparison between P. guineense 20 days after inoculation and P. guineense after 20 days of non‐inoculation with the nematode; gray circles—unchanged], and a Venn diagram of 135 differentially accumulated proteins (DAPs) after 20 days. (c) [GUA_20DAI/GUA_20DNI—comparison between P. guajava 20 days after inoculation and P. guajava 20 days after non‐inoculation with M. enterolobii, up (blue) and down‐regulated (pink); GUI_20DAI/GUI_20DNI—comparison between P. guineense 20 days after inoculation and P. guineense after 20 days after non‐inoculation with M. enterolobii, up (green) and down‐regulated (yellow).

FIGURE 5

Heatmaps together with interaction networks showing the up (A) and down (B) regulated proteins, comparing P. guajava 20 DAI and non‐inoculated with M. enterolobii (GUA_20DAI/GUA_20DNI) (a) and P. guineense 20 DAI and non‐inoculation with M. enterolobii (GUI_20DAI/GUI_20DNI) (b).