Methods to Quantify Biotic-Induced Stress in Plants

Abstract

Plant pathogens such as fungi, oomycetes, viruses and bacteria infect important crops and account for significant economic losses worldwide. Therefore, it is critical to gain insights into plant-pathogen interactions at the cellular and molecular level. The outcome of the interaction between plants and pathogens greatly differs depending on the species, strains and cultivars involved as well as environmental factors, yet typically results in stress for the plant, the pathogen or both. These biotic-induced stresses can be monitored using a wide range of techniques, of which some of the most commonly used techniques are outlined in this chapter. One widely observed feature of biotic stress in plants is the generation of reactive oxygen species (ROS) such as hydrogen peroxide (HO) and superoxide (O). We describe the quantification of hydrogen peroxide by 3,3'-diaminobenzidine (DAB) staining and luminol-based assays, and of superoxide by nitroblue tetrazolium (NBT) staining. Other techniques detailed here include measurement of callose deposition by aniline blue staining, evaluation of cell death by trypan blue staining and analysis of the loss of membrane integrity by monitoring electrolyte leakage.

AgroLux: bioluminescent Agrobacterium to improve molecular pharming and study plant immunity

“…HR has been quantified by binary scores (Kourelis et al ., 2020), by quantifying the percentage necrosis within the agroinfiltrated zone (Van der Hoorn et al ., 2001) or by using an HR index (Witek et al ., 2021). Cell death can be visualized sooner using trypan blue staining (Bach‐Pages and Preston, 2018), but this tedious procedure involves staining in phenol and de‐staining in chlorohydrate. Measuring leaked ions from leaf discs of agroinfiltrated leaves is another frequently used method to quantify the HR (Bach‐Pages and Preston, 2018), but this method is also laborious and often displays large variation.…”

“…Cell death can be visualized sooner using trypan blue staining (Bach‐Pages and Preston, 2018), but this tedious procedure involves staining in phenol and de‐staining in chlorohydrate. Measuring leaked ions from leaf discs of agroinfiltrated leaves is another frequently used method to quantify the HR (Bach‐Pages and Preston, 2018), but this method is also laborious and often displays large variation. Very recently, increased red fluorescence was reported for tissues undergoing the HR (Landeo‐Villanueva et al ., 2021), which is an interesting, complementary read‐out in comparison with the reduced bioluminescence displayed by AgroLux.…”

CLE14 functions as a “brake signal” to suppress age-dependent and stress-induced leaf senescence by promoting JUB1-mediated ROS scavenging in Arabidopsis

“…The fourth and fifth rosette leaves of 5-week-old plants were detached for NBT staining. The staining experiments were performed as previously reported (Fryer et al, 2002;Bach-Pages and Preston, 2018). A titanium sulfate standard curve method kit (Suzhou Keming, China) was used to measure endogenous H 2O2 content .…”

Metabolomic Evaluation of Ralstonia solanacearum Cold Shock Protein Peptide (csp22)-Induced Responses in Solanum lycopersicum

“…The leaves of 6 w old mature tomato plants were treated with csp22 and subsequently stained with a 3,3 -diaminobenzidine (DAB, Sigma, St. Louis, United States) solution to visualize and detect the presence of hydrogen peroxide (H 2 O 2 ). The protocol was performed with slight modifications as previously described (Bach-Pages and Preston, 2018). Briefly, the abaxial side of the leaves were treated with 500 nM csp22 by means of pressure infiltration and incubated for 30 min.…”